Protein Aggregation [1 ed.] 9781611221268, 9781617618154

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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

PROTEIN SCIENCE AND ENGINEERING

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PROTEIN AGGREGATION

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PROTEIN SCIENCE AND ENGINEERING

PROTEIN AGGREGATION

DOUGLAS A. STEIN

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Biomedical Books New York Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Protein aggregation / [edited by] Douglas A. Stein. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61122-126-8 (E-Book) 1. Proteins--Denaturation. 2. Protein folding. I. Stein, Douglas A. [DNLM: 1. Protein Folding. 2. Cell Aggregation. 3. Protein Conformation. QU 55.9] QP551.P69545 2010 572'.633--dc22 2010037892

Published by Nova Science Publishers, Inc. † New York

Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

Contents Preface Chapter 1

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Chapter 2

vii  Changes in Protein Structure under the Effects of Cryopreservation and Cryoprotective Agents N.G.Zemlianskykh  Molecular Chaperones and Proteases as Suppressors of Protein Aggregation in Gram-Negative Bacteria Joanna Skorko-Glonek, Dorota Kuczynska-Wisnik, Dorota Zurawa-Janicka, Ewelina Matuszewska, Donata Figaj and Barbara Lipinska 

Chapter 3

Native Functions of Amyloid Reeba S. Jacob, A. Anoop, Pradeep K. Singh and Samir K. Maji 

Chapter 4

Nucleation Mechanisms and Morphologies in Insulin Amyloid Fibril Formation Vito Foderà, Fabio Librizzi, Valeria Militello, Giovanna Navarra, Valeria Vetri and Maurizio Leone 

Chapter 5

On the Aggregation of Albumin: Influences of the Protein Glycation Philippe Rondeau, Giovanna Navarra, Valeria Militello and Emmanuel Bourdon 

Chapter 6

The Role of Conformational Domain Lability of Fibrinogen Molecules in Processes of Self-Assembly of Fibrin Monomers and Fibrinogen Aggregation M. A. Rosenfeld, V. B. Leonova and M. I. Biryukova 

1 

41 

79 

111 

139 

161 

Chapter 7

Two Faced Members of the Family: The Synucleins Andrei Surguchov 

179 

Chapter 8

Inclusion Bodies: A New Concept of Biocatalysts Neus Ferrer-Miralles, Mónica Martínez-Alonso, Antonio Villaverde and Elena García-Fruitós 

193 

Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

vi Chapter 9

Contents Comparative Study of Bovine and Ovine Caseinate Aggregation Processes: Calcium-Induced Aggregation and Acid Aggregation María Eugenia Hidalgo Manuel A. Mancilla Canales,   Cássia R. Nespolo, Anselmo D. Reggiardo, Estela M. Alvarez, Jorge R. Wagner and Patricia Risso 

Chapter 10

Protein Aggregation Xiaoyu Lu, Yan Wei, Weishan Wang and Rongqiao He 

Chapter 11

Yeast Protein Aggregates, Containing Chaperones and Glucose Metabolism Enzymes O. V. Nevzglyadova, A. V. Artemov, A. G. Mittenberg, E. V. Mikhailova, I. M. Kuznetsova, K. K. Turoverov and T. R. Soidla 

Chapter 12

Folding and aggregation features of proteins Oxana V. Galzitskaya and Sergiy O. Garbuzynski 

199 

223 

237 

267  281 

Index

283 

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Chapter Sources

Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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Preface Protein aggregation is the aggregation of mis-folded proteins, and is thought to be responsible for many degenerative diseases, such as Alzheimer's. This book presents current research from across the globe in the study of protein aggregation, including the processes of protein aggregation induced by freezing and lyophilization; functional amyloids; thermally induced aggregation of a model system protein - insulin; the aggregation of albumin; synucleins implicated in neurodegenerative diseases and some forms of cancer; yeast protein aggregates; and the folding and aggregation features of proteins. Chapter 1 – The long-term storage of various biological systems at low temperatures is possible due to the suppression of different types of molecular motions and an arrest of all metabolic and biochemical reactions. Nevertheless freeze-thawing and freeze-drying cause undesirable side effects, leading to cell destruction and loss of functional activity of proteins. Physical and chemical changes in environment in response to lowering temperature are related to the crystallization of liquid phase and concomitant increase in the concentration of all solutes in remained unfrozen solution, increment in viscosity, phase separation of constitutive components of system, buffering component crystallization with concomitant changes in pH, rise of an ice-aqueous interface and dehydration of macromolecules. Besides itself lowering temperature can cause cold denaturation of proteins. All those factors are potential stresses which alter proteins stability leading to loss of unique native structure and their functional activities. Stabilization of protein structure is achieved by application of cryoprotective agents, which are not restricted by a certain type of chemical compounds and presented by low molecular organic such as sugars, polyols, amino acids, methylamines as well as high molecular weight polymer compounds, including PEG, PVP, dextrans and HES. This review specifies the questions considering the mechanisms of structure impairment of protein, induced by cold denaturation, interactions protein macromolecules with ice crystals, dehydration, as well as the mechanisms of stabilizing effect of cryoprotective agents on protein behavior and modulation of macromolecular stability against physical and chemical stresses. In the review the processes of protein aggregation induced by freezing and lyophilization are also discussed. Chapter 2 – Protein misfolding is a detrimental and often deleterious phenomenon leading to a loss of functionality of a protein, formation of improper interactions or in many cases to aggregation. This process occurs efficiently especially under stressful conditions, e.g. exposure to high temperatures, oxidative or reducing agents, or as a result of certain mutations. Although bacteria are not endangered by numerous diseases associated with

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protein aggregation (unlike mammals), formation of aggregates represents a serious threat for these prokaryotic cells as well. In this review the authors present the current knowledge concerning the bacterial protein quality control systems. In the response to presence of misfolded proteins the specially dedicated defense systems are induced. They comprise molecular chaperones and proteases, whose function is to bind, prevent aggregation and refold polypeptides, or to degrade the irreversibly damaged proteins, respectively. In the cytoplasm of Gram-negative bacteria there are two major chaperone systems: (1) DnaK-DnaJ-GrpE and (2) GroEL-GroES. They collaborate with other chaperones: ClpB and small heat shock proteins IbpA and IbpB. The major cytoplasmic proteases are Lon, ClpAP, ClpXP, HslUV. The cytoplasmic protein quality control system is dependent on energy supply from ATP hydrolysis. The periplasm lacks the classical chaperone systems but it contains a group of proteins collectively named “folding helpers”, comprising also proteins with chaperone-like activity. They include SurA and Skp proteins engaged in folding of the outer membrane proteins. The major periplasmic protease is HtrA, responsible for degradation of misfolded proteins within the cellular envelope. It shows also a chaperone activity. Protein aggregation in bacteria represents also a serious problem in biotechnology as many heterologous proteins expressed in bacteria are deposited in form of inclusion bodies. Proper adjustment of the levels of molecular chaperones is be one of the solutions. Chapter 3 – Amyloids are highly ordered protein/peptide aggregates with cross-β-sheet rich structure. Amyloids are originally associated with many neurodegenerative diseases including Alzheimer's, Parkinson's and Type II diabetes. The natively structured or unstructured proteins adopt partially folded conformation and subsequently self-associates through nucleation dependent polymerization to form amyloid fibrils. These fibrils are very stable, resistant to proteases and to harsh environmental conditions. Recently, several studies have indicated that amyloid fibrils are also abundant in living organisms from prokaryotes to eukaryotes, where amyloids are evolved to perform native functions of the host. Such amyloids are termed as ‘functional amyloids’. Curli in E. coli and Het-s in Podospora anserina are well known examples of functional amyloids in bacteria and fungi respectively. Yeast prions do not cause cell death rather help the host to survive in certain environmental conditions. In mammals, Pmel17 forms amyloid inside the melanosome, where it is involved in skin pigmentation. Moreover, recent studies have suggested that peptide/protein hormones in pituitary secretory granules are stored in amyloid-like aggregates. In this chapter, the authors summarize the recent discoveries of functional amyloids, where amyloid fibrils are evolved for an organism's survival rather than creating only diseases. Chapter 4 – Aggregation processes, and in general the physical and chemical instability of proteins, are at the moment a major problem related to different scientific fields, spanning from biochemistry and biophysics to pharmaceutical and medical sciences. In fact, increased knowledge on protein aggregation may clarify different aspects related to several degenerative pathologies like Alzheimer's and Parkinson's diseases and type-II diabetes. In this chapter, the authors present and discuss their experimental results on thermally induced aggregation of a model system protein, the hormone insulin. This molecule is largely used in protein-based drugs and exhibits a great propensity to form amyloid aggregates with mechanisms similar to those of other disease-related proteins. Therefore, insights on insulin stability in adverse conditions as well as on mechanisms of fibril formation have a double relevance.

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Preface

ix

Using the scientific approach developed by their group in the last few years, the authors analyze the fibrillation kinetics of insulin as a function of the initial protein concentration paying particularly attention on the balance between different nucleation-elongation mechanisms and their effects on the final fibril morphologies. Using the fluorescence properties of amyloid sensitive dye Thioflavin T (ThT), static/dinamic light scattering and atomic force microscopy (AFM), the main role of secondary nucleation in determining the well-known exponential time course has been revealed. Specifically, the role of the early stable fibril surfaces and their ability in catalyzing the fibrillation reaction has been considered. Moreover, the fine investigation of the early stages of the process shows a pronounced stochasticity in the first aggregation events leading to an overall spatial heterogeneity in the formation of the early stable amyloid fibrils. These evidences are discussed and lead us to suggest a picture of interconnected events taking place at different stages in the process. Chapter 5 – Free radicals are a normal component of cellular oxygen metabolisms in mammals. However, free radical-associated damage is an important factor in many pathological processes. Aggregation, glycation, and oxidative damage cause protein modifications, frequently observed in numerous diseases. Albumin represents the most abundant circulating protein. Many epidemiological studies have established an inverse relationship between the level of serum albumin and the risk of death. Albumin is involved in several biological functions, including the regulation of oncotic pressure, and the binding and transport of many molecules. In addition, albumin displays potent antioxidant and free radical scavenging activities through the redox cycling of its free thiol and its ability to bind metal ions. Albumin constitutes a well-known protein capable of self-assembling in aggregates and also sensitive to glycative modifications, especially in cases of diabetes. In this review, the authors primarily report the different beneficial activities exerted by albumin, a multifunctional protein. They detail its importance in Human Physiology. Effects of aggregation modifications on albumin’s structure/function relationship are specified, bringing together recent insights on how aggregation processes in albumin can be affected by the protein glycation phenomenon. Chapter 6 – Fibrinogen is the major plasma protein of the blood coagulation system, molecular domain structure of which strictly corresponds to its main function – formation of insoluble fibrin. The mechanism of binding sites interaction localized on fibrinogen domains in order to construct protofibrils and fibrils proves to be well known. However, the role of conformational domain lability of fibrinogen molecules in processes of self-assembly of fibrin monomer and fibrinogen aggregation is still far from being completely understood. The article summarizes the data regarding fibrinogen domain D as being the most capable to local conformational rearrangements. Three-dimensional organization of intermediate soluble forms of fibrin-polymers in the presence of non-denaturizing urea concentration has been studied. Using the methods of dynamic and elastic light scattering combined with the analytic ultracentrifugation and viscosimetry it was shown that along with formation of traditional double-stranded protofibrils, in which fibrin monomer molecules by virtue of interactions between outer domain D and central domain E were arranged in a staggered overlapping manner, there was an alternative route of generation of equilibrium abnormal single-stranded, rod-like protofibrils formed in “end-to-end” fashion. It was concluded that local

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conformational transformation in regions of domains D remained the only way of the “end-toend” association. It was suggested that fibrinogen molecules could undergo local conformational transformation in regions of fibrinogen domains D during their incubation in a solution under the conditions close to the physiological ones. This results in accumulation of so-called “defective” molecules responsible for the process of fibrinogen aggregation. Molecules interact in “tail to tail” manner with formation of flexible single-stranded chain polymers. On attaining a critical length, polymers twist into a coil and aggregate to form branched clusters in which the segments are packed sufficiently dense to resemble strongly hydrated globular particles. It was suggested that damage of a native three-dimension organization of domains D causes an opening of the new reaction sites, which largely differed from the polymerization sites a and b “holes”. The effect of molecular “aging” of fibrinogen stimulated by pre-incubation in a solution both on the fibrin architecture and its ability to cross-link under the action of factor XIIIa has been studied. Fibrin generated from “defective” fibrinogen molecules had a coarser structure to be characterized by a higher mean mass-length ratio of the polymeric fibers compared to the native fibrinogen. Their data indicate that structurally modified fibrinogen molecules had more affinity for interchain cross-linking by formation of the ε/(γ-glu)lys isopeptide covalent bonds. The physicochemical mechanism of the fibrinogen molecular aging was demonstrated to be determined by processes that were identical to either its spontaneous oxidation (during incubation of this protein in a solution) or induced oxidation. Data on IR- and EPRspectroscopy methods obtained for oxidized fibrinogen fragments D and E make us believe the peripheral D-domains of fibrinogen as being the most sensitive to the free-radical oxidation. It is followed by the local structural conversions in regions of the domain D to expose reaction centers responsible for interaction in “end to end” fashion. The conjectural mechanism of conformational fibrinogen instability is discussed. Chapter 7 – α, - β- and γ-Synucleins are highly homologous small proteins implicated in neurodegenerative diseases and some forms of cancer. These proteins attracted the attention of many investigators, because of their role in human pathology. Certain soluble α-synuclein oligomers share a common structure with oligomers of other amyloidogenic proteins and peptides, for example, beta-amyloid (Aβ) and prion protein, implying a common mechanism of pathogenesis for several illnesses. γ-Synuclein can also form toxic protein inclusions, although the mechanism of its pathological action is not investigated in the same detail as αsynuclein. β-Synuclein prevents aggregation of α-synuclein and possesses neuroprotective effect. Here the authors give a brief overview of the structural features of synucleins which may explain why two members of the family (α- and γ-synucleins) can be easily converted into oligomeric toxic species, whereas β-synuclein has cytoprotective properties. Among important factors which cause transition of synucleins into pathological molecules are mutations, increased gene dosage (for α-synuclein), post-translational modifications (PTM) and binding to other proteins (for γ-synuclein). Oxidative conditions cause the formation of prefibrillar annular γ-synuclein oligomers which can induce aggregation of α-synuclein thus manifesting the property of antichaperone. Substances specifically preventing deleterious protein-protein interactions can be tested as potential drugs for the treatment of neurodegenerative diseases.

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Chapter 8 – Nowadays, many enzymes are used as catalysts in a wide range of bioprocesses in both chemical and pharmaceutical industries. Although the use of these biocatalysts has an enormous potential, the high cost of catalyst production is the most important bottleneck of the whole process. In this context, in the last decades, many enzyme immobilization strategies have been developed to favour the economical viability of enzymatic reactions in industrial processes. In this format, biocatalysts are then reusable and stable even under harsh conditions and can be tailored for different applications such as bioremediation, as biosensors and in the production of complex compounds, among others. Chapter 9 – The solubility of colloidal particles of bovine and ovine caseinate in the presence of calcium was studied by analyzing the colloidal particle size and the protein composition of casein colloidal aggregates remaining in suspension. A comparison between the behaviour observed for bovine and ovine caseinate was carried out. A two-step salting-out process, due to progressive Ca2+ binding to at least different two kinds of sites was observed for both caseinates. The precipitation curves were fitted and the affinity constants and binding site numbers were estimated with an equation based on the concept of Wyman’s linked functions. Ovine caseinate colloidal aggregates formed in the presence of calcium turned out to be less stable and quite bigger than the bovine ones. The binding of calcium to protein residues with some particular characteristics modifies not only the composition but also the conformational state of caseinates. An aggregation process at low caseinate concentration and an acid-induced gelation process at high protein concentration triggered by the hydrolysis of glucono-δ-lactone were also studied. The effects that variables such as temperature, protein concentration and GDL amount exerted on these processes were analyzed using spectroscopic-based methods and measuring the rheological properties of systems. The time required to initiate particles aggregation decreased in parallel with an increment of temperature, amount of GDL added and caseinate concentration. An increase in caseinate concentration or a reduction of temperature produced gels with a substantial rise in the storage modulus. Modifications of GDL/caseinate ratio did not resulted in significant changes in the rheological parameters determined. The kinetic of the aggregation and compactness degree of ovine caseinate aggregates formed at the end of the acidification process were different from those of bovine source. Acid-induced aggregation and gelation processes were also investigated in the presence of calcium concentrations where no precipitation occurs. The addition of calcium affects the kinetic of both processes and the final state of the aggregates or gels obtained. Consequently, the degree of compactness and average size of the aggregates and rheological properties of gels produced at the end of the acidification process depend on the calcium concentration added. Chapter 10 – Protein aggregation is the abnormal association of proteins intracellulary or extracellularly. In general, it is the posttranslational aftermath of a gene mutation or the effect of environmental stress or aging, causing protein misfolding and undesirable intermolecular interactions between misfolded proteins which, in turn, aggregate together to form either fibrils or amorphous deposits. Chemical modifications, such as phosphorylation, glcyation (glucosylation, ribosylation), and hydroxymethylation (with formaldehyde), play an important role in the formation of proteinaceous aggregates. Amyloidosis is the accumulation of insoluble proteinaceous aggregates in vivo and has been implicated in many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). As such, the human central nervous system seems to be especially vulnerable to the proteotoxic nature of protein aggregation. The tendency for

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insulin to aggregate and form fibrils has serious implications for the production, storage and delivery of the protein, and persists to be a problem in new delivery systems currently under development. This article briefly reviews the current understanding of protein aggregation. Chapter 11 – Three groups of proteins associated with misfolded protein depending aggregates were identified in Saccharomyces cerevisiae cells by using a new approach: comparative analysis of crude lysate pellets of isogenic yeast strains differing by their prion composition or adenine biosynthesis pathway characteristics. 2D electrophoresis followed by MALDI analysis of a recipient [psi-] strain and of [PSI+] cytoductant permitted identification of 53 proteins whose aggregation state depended on prion content or red pigment accumulation in yeast cells. Further studies allowed identifying an overlapping group of 38 proteins whose aggregation state responded to a shift of prion(s) content and also a rather similar group of more than 40 proteins whose aggregation state depended on accumulation of red pigment. In all these cases nearly one half of the identified proteins belonged to a functional group of chaperones and enzymes involved in glucose metabolism. Notable were proteins involved in oxidative stress response and in translation. The prion dependent group also contained a proteinase. These results are comparable with recent literature data on various misfolded proteins containing aggregates in yeast cells. Being not dependent on cloned heterologous genes, the authors approach permits a conclusion about universal presence of glucose metabolism enzymes in such aggregates. Most of the identified proteins, although behaving like prions in some experiments (for example, being “transmittable” by cytoduction), seem to be just amyloid-associated and mobilized to pellets in response to presence of prion fibrils. Their model experiments demonstrate that red pigment binds insulin fibrils and blocks their interaction with Thioflavine T. This allows concluding that red pigment impedes mobilization of some prion-associated proteins to prion-containing aggregates and so makes them to appear as pigment depending ones. Also there are some proteins (e.g. Sod1p and Cus1p) that themselves can be “clients” of a hypothetical prion generation pathway dealing with not NQ-rich proteins in yeast. Chapter 12 – In addition to "normal," native protein structure, some proteins can also form alternative, misfolded structures. During the past years, it has been shown that some diseases are connected with protein misfolding and the formation of insoluble aggregates called amyloid plaques. For some proteins which are capable to form amyloid structures, those regions which are important for amyloid formation are already experimentally outlined (they are called amyloidogenic regions). In these proteins, the authors predicted those residues that are important for "normal" folding (that is, which are involved into the folding nucleus of the native structure) and compared them with amyloidogenic ones. The average of the predicted Φ-values (which reflect the degree of involvement of the amino acid residue into the folding nucleus) over 12 amyloidogenic regions (of 7 globular proteins in which amyloidogenic regions are now localized experimentally) is significantly greater than the average Φ-value averaged over residues outside amyloidogenic regions. This demonstrates that amino acid residues in amyloidogenic regions in average are more included into folding nucleus than amino acid residues from non-amyloidogenic regions. In total, 8 of 12 amyloidogenic fragments are located in the folding nuclei. This is an indication that amyloidogenic regions are typically incorporated into the native structure early during its formation (not later than at the rate-limiting step of a "normal" folding process).

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Chapter 1

Changes in Protein Structure under the Effects of Cryopreservation and Cryoprotective Agents N.G.Zemlianskykh* Institute for Problems of Cryobiology and Cryomedicine of NAS of Ukraine, Kharkiv, Ukraine

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Abstract The long-term storage of various biological systems at low temperatures is possible due to the suppression of different types of molecular motions and an arrest of all metabolic and biochemical reactions. Nevertheless freeze-thawing and freeze-drying cause undesirable side effects, leading to cell destruction and loss of functional activity of proteins. Physical and chemical changes in environment in response to lowering temperature are related to the crystallization of liquid phase and concomitant increase in the concentration of all solutes in remained unfrozen solution, increment in viscosity, phase separation of constitutive components of system, buffering component crystallization with concomitant changes in pH, rise of an ice-aqueous interface and dehydration of macromolecules. Besides itself lowering temperature can cause cold denaturation of proteins. All those factors are potential stresses which alter proteins stability leading to loss of unique native structure and their functional activities. Stabilization of protein structure is achieved by application of cryoprotective agents, which are not restricted by a certain type of chemical compounds and presented by low molecular organic such as sugars, polyols, amino acids, methylamines as well as high molecular weight polymer compounds, including PEG, PVP, dextrans and HES. This * N.G.Zemlianskykh Senior scientist; Department of cryocytology, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine 23, Pereyaslavskaya str., 61015, Kharkiv, Ukraine [email protected]

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N.G. Zemlianskykh review specifies the questions considering the mechanisms of structure impairment of protein, induced by cold denaturation, interactions protein macromolecules with ice crystals, dehydration, as well as the mechanisms of stabilizing effect of cryoprotective agents on protein behavior and modulation of macromolecular stability against physical and chemical stresses. In the review the processes of protein aggregation induced by freezing and lyophilization are also discussed.

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Introduction In biothechnonlogical elaborations applying in medicine, pharmaceutical and nutritive industries the low temperature techniques are widespread for storage of biological subjects of different level of organization [1,2]. Cryopreservation is the dominant technology that maintains ex vivo biological structure and function of living systems by freezing to ultralow temperatures and provides long-term storage of macromolecules, cells and tissues. The stabilization of biological systems under cryopreservation conditions is favorable due to the suppression of different types of molecular motions and as a consequence an arrest of all metabolic and biochemical reactions in response to lowering temperature. Freeze-drying (lyophilization) is also applied for long-term storage of biological subjects. As distinct from cryopreservation, lyophilization is successfully used mostly in pharmaceutical protein formulations, as only several types of cells, essentially prokaryotes [3-5], maintain the acceptable level of viability after the freeze-drying proceeding. Attempts of usage of freezedrying for storage of mammalian cells proved to be quite inefficient and the results of research projects in this field are still far from the possibility of their putting to use. In particular, statements concerning the preservation of red blood cells in a dried state proved to be contradictory [6-9]. Nevertheless in several reports it was assumed that frozen-dried human platelets are nearly ready for clinical trials [10-12]. Unprotected freezing is normally lethal for cells [1,2,9] and accompanied by partial or complete inactivation of proteins in pharmaceutical formulations [13-15]. The most essential source of cryoinjuries of biological structures is related to ice crystal formation that destroy cells through an immediate mechanical action [16] or through secondary effects of alteration of liquid phase composition [17,18]. The two mechanisms of cryoinjuries are important and their relative contributions depend on a cell type, cooling rates and warming ratеs. Actually, upon decline in temperature primarily water crystallization occurs in an extracellular medium and that determines in many respects the following advancement of events. However, cell alterations arising from extracellular water crystallization, distinct from damages related to ice formation inside cells, which often proved to be lethal [19,20]. Consequently for successful cryopreservation it is indispensable to provide conditions which permit to avoid the intracellular ice formation during freeze-thawing. Whereas temperature impacts essentially the rates of water transfer across the plasma membrane, the water efflux and influx during cooling and thawing accordingly are dependent on the rate of temperature change in a system. Generally cell injuries under freeze-thawing can be explained relying on principles of two factors’ theory of cell cryoinjuries [21] which permit to describe the changes proceeding in environment during cooling and freezing and understand stresses that induce biological structure alterations. While water is freezing in extracellular medium, an increase in solute concentrations in the retained nonfrozen liquid fraction of extracellular medium is under

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Changes in Protein Structure under the Effects of Cryopreservation …

3

progress. Arising from those events, difference of chemical potentials across the cell membrane provides the driving force for the efflux of water from the cell. If the cooling rate is sufficiently slow, the movement of water across the membrane will maintain the intra- and extracellular compositions close to chemical equilibrium. Thus, at slow cooling rates the cell has time to respond by exosmosis resulting in cellular dehydration, volume reduction and increase in intracellular concentration over time. The injuries of biological structures upon a slow colling are related to the negative influence of “solution effects” [17,18]. When cells are cooled rapidly, formation of ice in external solution and concentration of extracellular solutes occur much faster then the efflux of water from the cell. As a result cytoplasm becomes increasingly cooled below the freezing point (supercooled) with an associated increase in probability of intracellular ice nucleation [20,21]. Therefore the combination of these two factors, solution effects and intracellular ice formation, determines the optimization of cooling rates for each cell type. Nevertheless absolute survival of cells upon freezing without of cryoprotectant agents remains extremely low. Cryoprotectants generally contribute to increase in common concentration of all solutes in a system and decrease in amount of ice crystal formation at the given temperature. Nevertheless, it has recently become clear that the cryoprotectant effects to biological structures are more complex than it was expected earlier and are not limited by colligative properties [23,24]. The injuries of biological structures upon freezing are determined not only by peculiarities of integrated cell reactions based on osmotic and diffusion processes but also largely by biomacromolecule instability, in particular, proteins which react by structure alterations to low temperature and concomitant changes in physical and chemical environmental parameters [13,25]. During freezing physical environment around proteins changes essentially that leads to the development of stresses that impact protein stability. Freezing of protein solution and cells may result in irreversible protein aggregation and severe loss of catalytic activity of enzymes [26], breakdown of membrane transport functions [27] and collapse of cytoskeleton network [28]. All those entail reduction in cell viability or loss of therapeutic value of pharmaceutical proteins. As causes responsible for structural disturbances of proteins one should list some main factors: low temperature as a cause of cold denaturation; ice formation connected with appearance of ice-aqueous interfacial area; solute concentration increment; probability of buffer components’ crystallization accompanied by large change in pH increase in viscosity of unfrozen concentrate; possibility of phase separation depending on the composition of formulation; protein-solutes and protein-protein interactions leading to protein aggregation . The different cryoprotectant agents are able to a different extent to prevent structural disturbances of proteins and to interfere their aggregation under stress conditions of freeze-thawing and freeze-drying [29,30]. This review considers the main factors affecting the protein structure upon freezing and in a part upon lyophilization, namely, protein cold denaturation, interaction protein with ice crystals, protein dehydration, as well as represents insights into mechanisms of protein stabilization under cryoprotectant agent effects. In the review the processes of protein aggregation observed after freezing and lyophilization are also discussed.

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Cold Denaturation of Proteins The phenomenon of cold denaturation is related to breakdown of compact native unique three-dimensional structure of protein macromolecule during decrease in temperature. Theoretically cold denaturation should be a universal property for all proteins reflecting interaction between water and protein molecules at low temperature. Cold denaturation has been investigated for a number of proteins, including β-lactoglobulin [31], chymotrypsinogen [32], lactate dehydrogenase [33], myoglobin [34], phosphoglycerate kinase [35], ribonuclease [36], staphylococcal nuclease [37], monomeric λ repressor [38], CheY [39], the human fibroblast growth factor [40], barstar [41], ubiqutin [42]. Although, cold denaturation is considered as a general property of proteins this event can be observed only at sufficiently low temperature, as a rule, below water freezing point. That creates many experimental complications for analysis of structural and thermodynamic changes of protein macromolecules during cold denaturation related to certain limitations of technique possibilities. To overcome these complications proteins are primarily exposed to destabilization conditions that permits to observe cold-induced unfolding above water freezing point. For this purpose one usually applies different approaches which include chemical denaturants (mostly, guanidinum hydrochloride or urea), alterations of pH and hydrostatic pressure as well as site-directed mutagenesis that causes their structural destabilization. In general, cold denaturation is caused by the very specific and strongly temperature-dependent interactions of protein groups with water [43,44]. Actually protein is thought to be stable within a certain range of temperatures and both lowering and increasing temperature lead to thermodynamic instability of proteins. It implies that beyond limits of the physiological range at higher and lower temperatures denatured states of proteins become more favorable and unfolding are accompanied by a decrease in free energy (∆G). Generally, temperature dependence of ∆G for any model protein should have a convex form with maximum positive value relevant to temperature of physiological activity and indicates the presence of two-phase transitions when ∆G(T)=0, which correspond to heat and cold denaturation. Cold denaturation is accompanied an increase in surface area of nonpolar groups exposed to water, which normally are buried inside core domains of macromolecules, due to the weakening of hydrophobicity at low temperature and an increase in amount of water molecules forming ordered structures, which lead to negative changes in water entropy and energy. It is experimentally known that both enthalpy and entropy increase upon heat denaturation. Cold denaturation is accompanied negative changes both in enthalpy and entropy. The negative enthalpy changes for cold denaturation seem to be counterintuitive because denaturation leads to loss of the protein intramolecular energy. The negative entropy change at cold denaturation also seems to be counterintuitive because of a large gain in protein conformational entropy. In order to understand the counterintuitive behavior and the thermodynamic properties of cold denaturation of a protein many simulations were performed with emphasis on the hydrophobicity by constructing a minimal model for protein-water system [45-47]. For example [45,48], there is a simulation providing microscopic picture for cold denaturation in terms of changes in hydration according to which at low temperature water molecules infiltrate the folded protein in order to passivate the dangling water-water hydrogen bonds (H bonds) found in shell water. As at low temperature hydrophobic contact

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are destabilized an ordered layer of water forms around the exposed hydrophobic groups of polymer chain with separation them by layer of solvent stabilizing the cold denatured state. Besides, this approach can explain to some extent enthalpy and entropy changes of the entire system during cold denaturation. Generally, as a nonpolar solute is inserted in water at the given positive temperature its molecules tend to cluster to reduce the amount of shell water in a system because shell water has a higher free energy than the bulk water, which means the hydration free energy is positive. Actually, the tendency of nopolar solutes to cluster is known as the hydrophobic interactions. Since shell water is more ordered and forms more H-bonds than the bulk water, the clustering of solutes increase the enthalpy and entropy of a system. Thus hydrophobic interactions are stabilized by entropy and destabilized by enthalpy (entropically driven stabilization). During cooling the average strength of the interactions between atoms increase as thermal energy decreases but hydrophobic interactions weaken with lowering temperature. As a result upon cooling both the stabilizing effect of entropy and destabilizing effect of enthalpy enhance. The increase in entropy indicates that the difference between shell and bulk water increases because shell water becomes more ordered and forms more H-bonds than bulk water. However the entropic and enthalpic terms do not change at the same rate. The enthalpic penalty increase faster upon cooling and as a result free energy of entire system becomes negative. Denatured states of protein induced by heating and cooling from the temperature, at which it is stable, are distinguished by thermodynamic characteristics and could differ structurally. That is seen by the example of bovine β-lactoglobulin, heat and cold denaturation parameters were characterized by scanning callorimetry, circular dichroism (СD) and NMR spectroscopy [31]. The most convenient condition for observation of heat and cold denaturation of β-lactoglobulin is a buffered solution containing 4.0 M urea, where the protein exist as a monomer and completely reversible after either heat or cold denaturation. Structural changes in the protein were observed by the parameter G of NMR-spectrum which measures the efficiency of the spin diffusion and reflects the compactness of macromolecules, correlating, to some extent, with conformational entropy. The monotonic decrease in G parameter with rising temperature indicates an increase in a different kind of motions in the protein: motion of specific side chains, sequential motions and motions of the molecule as a whole. In contrast, the decline in temperature, with a concomitant decrease in dissipative forces, leads to rise in G-parameter, reflecting the increase in rigidity of the regular conformation in the native protein. The differences between parameters Gal and Gar (aromatic and aliphatic groups) for β-lactoglobulin correlate with structural integrity of the unfolded molecule. Disruption of native β-lactoglobulin structure during heating is accompanied by drawing together of all motions of constituent structure parts of the molecule, correlating with decrease in G-parameters. The conformational entropy becomes large and similar in magnitude for aromatic and aliphatic groups of the molecule. The disruption of native structure during cold denaturation does not lead to convergence of Gal and Gar parameters, as it was observed in the case of the heat denaturation. The parameters Gal and Gar achieve the maximal values at the temperature of cold denaturation but the difference between the values of Gal and Gar remains unchanged at the same time (31). Then these functions decrease sharply but below this temperature do not change significantly in magnitude in response to further decrease in temperature remaining more or less continual. Thus analysis of the NOE spectra of this protein shows, that changes in the spin diffusion of β-lactoglobulin after

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disruption of the unique tertiary structure upon cold denaturation are much more substantial, than those upon heat denaturation. In addition, it was supposed that cold denaturation is not a two-state transition that is some stable intermediates are present during the transition between the native and denatured states of β-lactoglobulin, while heat denaturation of the protein in over 4.0 M urea represents a two-state transition without stable intermediates or with an extremely small population of intermediates. Besides, it is suggested that cold denaturation involves disruption of intermediate simultaneously with disruption of tertiary structure and is accompanied by substantial changes in secondary structure, heat capacity and efficiency of spin diffusion. As a result enthalpy of this transition shows deviation from that expected for a two-state model. The heat denaturation leads to disruption of mostly tertiary structure and this process is not accompanied by substantial changes in heat capacity, ellipticity or efficiency of spin transision. Finally, it was concluded that denatured β-lactoglobulin exhibits extensive residual hydrophobic and hydrophilic interactions. Although the structure stabilized by these interactions is much less fixed than in the native one it, however, displays a flexibility and rigidity (parameter G) similar to that of the native molecule. Relying on the difference parameter G and the data of heat capacity change it was supposed that in cold denatured βlactoglobulin the network of residual interactions in hydrophobic and hydrophilic regions of the molecule is more extensive than that after heat denaturation. This suggests that upon cold and heat induced unfolding, the molecule undergoes different structural rearrangement, passing through different denaturation intermediates. Cold denaturation of β-lactoglobulin can be considered as a two stage process with a stable intermediate. It is worth noticing that a similar equilibrium intermediate can be obtained at 35oC in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of this intermediate. However, these processes differ in cooperatives, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption over cooling is accompanied by cooperative changes in heat capacity, whereas heating only changes the heat capacity gradually. Cold denaturation state of equine β-lactoglobulin as well as bovine β-lactoglobulin [49] is distinguished from the heat denatured or acid denatured ones. Nevertheless in the presence of 2.0 M urea a cooperative unfolding transition was observed both with increasing and with decreasing temperature. The heat and cold denatured states of equine β-lactoglobulin have substantial secondary structure but lack persistent tertiary packing of the side-chains. The investigation by angle X-ray scattering and analytical ultracentrifugation indicated that equine β-lactoglobulin assumed an expanded chain-like conformation in the cold denatured state. The CD spectrum shows a significant amount of secondary structures including non-native αhelices. In contrary, the secondary structure of bovine β-lactoglobulin is largely disrupted in the cold denatured state which can be observed in 4.0 M urea [31,50]. This discrepancy may be attributed to the difference in protein sequences. Thus the secondary structure of bovine βlactoglobulin is less organized than that of equine β-lactoglobulin during cold denaturation, nevertheless it remains to be clarified whether this reflects the structural difference in the cold denatured states or the difference in stability of the cold-denatured states. Comparison of the heat- and cold-denatured states shows that the heat denatured state of equine β-lactoglobulin is similar to the molten globule state and that the cold denatured state

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of equine β-lactoglobulin assumes a helical and expanded conformation. The fact that the molten globule is transformed to the expanded cold-denatured state at a lower temperature indicates that molten globule structure of equine β-lactoglobulin is stabilized by numerous hydrophobic interactions and there is possibility that some helices formed in the colddenatured state are disrupted and/or transformed to other conformations by long-range hydrophobic interactions with increasing temperature. Actually, the discrepancy between heat and cold denatured states may be caused by a variety in the factors responsible for structural stabilization of proteins. It is suggested that cold denaturation is attributed to the weakened hydrophobic interactions at low temperature while heat denaturation is generally due to the enhanced chain mobility at high temperature [43]. Thus the comparing the conformation differences between heat and cold denatured states may shed light on the hierarcy of interactions responsible for the structural stabilization of a protein. For example, cold denaturation of yeast phosphoglycerate kinase indicated independent unfolding of two domains whereas heat denaturation was highly cooperative [35,51] that gives important information on the structural organization of this protein. On the other hand, whereas cold denaturation is an inherent thermodynamic property of proteins caused by a large heat capacity difference between the native and denatured states, the both states could be considered as the same macroscopic state [52]. The conformational similarity between the cold and heat denatured states has been actually shown for several proteins [53,54]. With the yeast frataxin Yfh1, which possess a very low heat denaturation temperature, it was exhibited [55], that the recorded NMR spectra of the protein at -5 and 45 o C are collapsed and in agreement with a completely unfolded states. The small difference at low and high temperature can be easily accounted for the different exchange rates of the amide protons at different temperature. According to the thermodynamic parameters it was concluded that disruption of the hydrophobic core was exactly paralleled by a decrease in the secondary structure content, although cold denaturation has “thermodynamic anomalies” and both ∆H and ∆S are large and negative for this transition. Assuming that cold and heat denaturation are the same macroscopic states then the cold unfolding of globular proteins can be well described by a two-state transition between native and denatured states, thus implying an all-or-nothing single-step mechanism for the collapse of protein tertiary structure [52]. For example, cold unfolding of apomyoglobulin (apoMG) is tighly related to the stability of the hydrophobic core formed by the A-, G-, and H-helices. At a temperature below the cold denaturation point, the A-helix detaches from the G- and Hhelices and the unfolding occurs. In addition, the appearance of the pre-transision prior to unfolding was suggested to correspond to the creation of a relatively conformationally loosed native state [52] and the all-or-nothing mechanism has been accepted. Nevertheless cold unfolding process does not imply a complete structural loss in proteins. Analysis of simulations of apoMG behavior upon cold denaturation [52] has shown that an enhancement in hydrophobic group solvation at lowering temperature causes packing changes at the waterprotein interface, as water penetrates interstitial spaces at the interface. The fluctuations of some protein atoms increase with decreasing temperature. The internal fluctuations are a direct result of the peripheral penetration of water that can lead to the development of instability within the protein core and eventual breakdown of tertiary associations. As such breakdown of the protein would occur in steps, depending on the relative stability of individual hydrophobic protein core contacts. Probably, in the case of a small protein with a single core region, such as apoMG, the unfolding would take place in a single step.

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Observations of single [53-53] or multiple step [56] denaturation events are therefore compatible with this interpretation of a cold denaturation mechanism. The pre-transition prior to unfolding creates a conformationally loosed, more highly fluctuating state. Application of different approaches, in particular, chemical denaturants to disturb protein structure in order to observe cold denaturation at higher temperature tend to distort the energy landscape of the native state and can potentially obscure features unique to cold denaturation. To avoid these issues it was proposed to encapsulate a singe protein molecule in a reverse micelle dissolved in a low viscosity fluid, where molecular reorientation and slower spin-spin relaxation improve NMR spectroscopic performance [57]. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that proteinsurfactant interactions are negligible and allow the direct observation of multi-state coldinduced unfolding of the examining protein ubiquitin [57]. It has been recognized for a very long time that a protein of even modest size has a number of potential conformations and that can be related to denaturation behavior of protein. The approach permitting to differentiate cooperative substructure of proteins is grounded on the fundamental thermodynamic parameters, which predicts dependence of various types of interactions, which stabilize protein structure, on temperature. Hydrophobic interactions are generally characterized by a large heat capacity while polar interactions are generally close to zero when they disrupt in water. The states with different heat capacity values underlying protein stability can potentially be distinguished and significantly populated during cold-induced disassembly [56].This is a distinct contrast to high temperature unfolding where, effective two-state behavior is generally observed and careful analysis can only infer the presence of intermediates [58]. Cold-induced unfolding of ubiquitin encapsulated in reverse micelles was found to be highly noncooperative, in distinct contrast to its apparent two-state thermal unfolding. A different result was obtained if a high salt buffer was used to screen surfactantprotein charge-charge interactions [59]. It was claimed that cold-induced unfolding of encapsulated ubiquitin occurred in a two-state manner in the presence of 1.5 M NaCl and implied that multi-state unfolding was an artifact of protein-surfactant interactions. But that result [59] was not confirmed in [57] and cold-induced unfolding of encapsulated ubiquitin proved to be inherently multy-state. The apparent disagreement in results of these two studies could be explained in terms that several quantitative criteria were not considered. Probably one did not take into account that in a high ionic strength a high chloride concentration would stabilize the protein [60] against cold-induced unfolding and ubiquitin encapsulated in a high ionic strength buffer in charged surfactant AOT cold-denatures at a slightly lower temperature than when encapsulated in a neutral surfactant [57]. The low temperature behavior of ubiquitin under variety of conditions have been reexamined [57] and the cold-induced unfolding process was confirmed to be highly non-cooperative and decidedly multi-state and protein-surfactant interactions played a minor role and did not interfere significantly. Presently it is recognized that accurate analysis of heat and cold denaturation processes has potential to unveil invisible till now aspects of protein dynamics and stabilization. The difference between the cold- and heat-denaturated states, especially for small apparent twostates proteins, becomes feebly distinguished because both of them lack any significant buried hydrophobic surfaces. The main difference results from the residual preference for their secondary structure. The folding kinetics is expected to be a more sensitive distinguishing property between these two states [61]. Thus changes from non-exponential to exponential folding kinetics, while the temperature of phosphoglycerate kinase is lowered, have been

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attributed to a decrease in energy landscape roughness in the cold-denaturated state, which does not occur in the heat-denaturated one [62]. The folding kinetics of cold- and heatdenaturated states of a small 80-residue 5 helix bundle protein λ6-85 are identical within measurement uncertainty under the condition if a correction for solvent viscosity is made [63]. The study of folding/unfolding kinetics of a mutant variant protein λ6-85 showed that relaxation rates corrected for viscosity were identical for relaxation between the native and either the cold- or heat-denaturated states, as long as experiments were compared at identical folding free energies. Cold and heat denaturation processes are “symmetrical” for this mutant of the protein λ6-85 as far as the folding barrier height is concerned. The subtle differences between the two ensembles of the λ6-85 could be related to that the hydrophobic effect controls barriers, and that the two unfolded ensembles of this small helical bundle are identical in terms of exposed hydrophobic surfaces. The investigation of 15 different mutants of λ6-85 permitted to divide them into 2 groups, one of them has a turnover in folding rate, other one has anti-Arrhenius folding behavior. The peculiarities of the two group behavior were attributed to a competition between hydrophobic contact formation, which should follow the strength of the hydrophobic effect and have a turnover at intermediate temperatures, and secondary structure formation, which was greatly enhanced at lower temperatures in the alanine-containing molecules. The triple mutant, which was the subject in this study, falls into limit of destabilization secondary structure, so collapse controls the formation of the native state. On the other hand, it is expected that mutants obeying anti-Arrhenius folding behavior will have heat and cold-denaturated ensembles distinguishable by their folding rates. At this rate the issue of how close to equivalent the heat- and cold-denaturated ensembles may be addressed to a simple model that combines the hydrophobic contact formation and the temperature dependence of helix-coil transition (secondary structure formation) which was presented in [63] referred to H.Kahara and co-workers. Collapse due to hydrophobicity is symmetric with respect to the temperature of highest collapse propensity, and is slowed down equally at lower and higher temperatures. They yield relatively symmetrical population profiles for heat- and cold-denaturated states. The helix-coil transition is only weakly cooperative, and changes much more slowly as a function of temperature, with increasing helix at low temperature. When the two tendencies are superimposed, an asymmetric denaturation profile is obtained. The relative contribution of these two effects determines the asymmetry of the two phase transitions, and hence whether hydrophobicity or secondary structure formation controls the kinetics. Most proteins studied under cold denaturation process belonged to either α- or α+βstructural classes. Cold-induced unfolding of aponeocarzinostatin (apoNCS), which is all-βsheet protein, gives interesting information in terms of temperature dependence of denaturation process on structural peculiarities of proteins. Actually a wide range of unfolded non-native states has been found in several proteins [64]. These non-native states can vary from partially structured to unstructured random coils. The apoNCS under cold-induced unfolding exists in a partially structured state: tertiary structure of apoNCS disrupted, but secondary structure remains folded. The partial loss of the highly compacted structure upon cold-induced unfolding is not a rare phenomenon for α-helical-based proteins. Similar observation has been reported for the molten globule state, in particular, for cytochrome c [66], in which the secondary structure is formed, but the tertiary structure fluctuates considerably. Partially unfolded state of myoglobulin at cold-induced denaturation is also characterized by a persistent amount of secondary structure, whereas rigid tertiary structure is

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lost [67]. A molten globule-like intermediate can be also formed by a β-sheet protein [68,69]. Computational theories on CD of protein folding intermediates suggest that it is possible for an all-β-protein in a molten globule state to retain all or almost all of the individual units of secondary structure β-sheets and probably β-hairpins [70,71]. These secondary structure units fluctuate with respect to each other. In most cases the β-sheets are still rather local and not assembled from very remote segments in the sequence. The common types of sheets are likely to maintain their integrity under mild conditions leading to molten globule formation. So it was also assumed that the β-sheet apoNCS formed an unfolding intermediate by losing its near-UV CD signal (tertiary structure) but without losing much of its signal in the far-UV CD (secondary structure) [72]. Since cold-induced unfolding is mainly caused by interactions of protein groups with water the exposure of internal surface to water can be under control of ligands. The protein NCS is a good demonstration of chromophore effect on the protein stability [72]. Labile enediyne chromofore which is highly stabilized by the protein is able to protect the protein against cold-induced denaturation, but not against the heat-induced unfolding. The tightly bound enediyne chromofore, occupies the central cavity in the compact native structure of NCS protein, and diminishes the changes in exposing internal protein groups to water, efficiently reducing the hydration effect. As for the heat-induced unfolding, it is mainly initiated by a large increase in entropy at high temperature, the chromophore cannot act as an effective stabilizer for the protein, which is disrupted by the heat-induced molecular motion. Summarizing data on the structural changes of proteins caused by cold denaturation one can suggest that such rearrangements in macromolecules may create non-native states, which possess a high prone to aggregation tendency and there are many experimental confirmations related to aggregate formation induced by lowering temperature [67,73,74].

Disturbance of Protein Conformation Affected by Ice Formation Essential progress in study of structural changes of proteins under the conditions of a liquid phase crystallization was acquired due to development of new experimental approaches which are grounded on the changes of fluorescence and phosphorescence parameters of tryptophan residues of proteins or fluorescence characteristics of ANS (8-anilino-1naphthalene sulfonate) probe in frozen solution of proteins. The phosphorescence emission of tryptophan (Trp) residues can serve as a monitor of the polypeptide dynamical structure reflecting conformational changes of proteins in response to variations in conditions of the solution. At first the study was performed for azurin, a small protein with the known structure [75-79]. It was shown that [80] at -12 oC, when supercooled solution solidifies, phosphorescence lifetime of Trp τ dropped by ~ 30 folds that indicate a dramatic gain in the polypeptide flexibility. This phenomenon was observed with other examined proteins, namely, monomer protein ribonuclease T1, dimers LADH and AP, tetramers – GAPDH and LDH. Ice-induced decrease in τ is attributed to a general and extended loosing of native fold or a partial unfolding that involves the rigid protein domains hosting the phosphorescence probe. To validate what factors of solution conditions during ice formation is responsible for protein structural change many parameters were tested.

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Proceeding from the given experimental conditions a number of such parameters which might underlay a sharp decrease in τ to response of ice formation could be excluded directly as determinant mechanism of unfolding of protein molecule. The long lifetime in supercooled solution, τo, proves that low temperature per se is not a destabilizing factor. A drying effect (the low activity and peculiar properties of interstitial water) is also discounted because hydration water crystallizes only in part and at much lower temperature. Besides protein dehydration yields enhanced rigidity as evidenced in the generally large value of τ for fully desiccated protein powders [81]. The effect of pH changes is also excluded due to experimental data with varying buffer systems. Protein-protein interactions are excluded by the results of absence of dependence of lifetime phosphorescence profile on the examined protein concentration, and in addition at even larger concentrations there is a distinct attenuation of the decrease in τ. The same was concluded in respect of possible interaction of protein with buffering salts or NaCl in the unfrozen liquid, although some additives can modify the lifetime response to freezing, τo is only moderately sensitive to molar quantities of these solutes. Thus it was concluded that the main source of disruption of protein structure at solution solidification is related to interactions of protein molecules with ice crystals and these findings are based on serious experimental evidences. The interaction of protein with ice crystals can occur as adsorption process or physical distortion of protein globule confined in the intergranular space of ice crystals by anisotropic compression [82]. In the first case the protein should be distributed between liquid and solid phases and the extent of adsorption will be determined by the volume fraction of liquid water, VL, and the surface area of ice. In the second case the perturbation is expected to be when the average size of intergranular spaces is comparable with or smaller than the protein molecular volume. Adsorption should manifest itself as gradual process, whereas purely mechanical effects of ice formation would exhibit a more-or-less sharp threshold at some specific VL, a threshold governed by the molecular weight of the protein. The finding that substantial changes in τ occur even with large volume of unfrozen liquid (Vl~10%) testifies to the mechanism of protein absorption to ice and tend to rule out mechanical distortion of the protein globule. Besides, the character of protein-ice interactions are affected by the texture of ice because, for any VL, the number and size distribution of ice crystals determine the total ice surface area and the actual size of intergranular spaces. The texture of ice depends, among other factors, on the cooling rate and on aging. Slow cooling and prolonged maturing promote the formation of small amount of large ice crystals. Smaller perturbation of protein structure is associated with the reduction in ice surface area, which is confirmed by data related to studying the effect of cooling rates on the alteration of parameter τ. Slow cooling yields a smaller perturbation of τ that is in agreement with decrease in protein absorption. Thus study of Trp phosphorescence lifetime changes provided a direct evidence that [80] solidification of water led to essential perturbation of globular fold in all the proteins examined. From the drastic shorting of phosphorescence lifetime of Trp residues buried in the rigid core of these macromolecules it was inferred that in ice the internal flexibility was greatly enhanced as a characteristic of partially unfolded polypeptides or the loss of tertiary structure and as a possibility of the formation of molten globule state [83,84]. The perturbation may drive from the adsorption of the macromolecules to the liquid/solid interface. The limitation of this approach for analysis of protein structural changes is related to the fact that this method only provides indirect information on the structure of the macromolecule and only concerns alterations in specific sites in the protein interior, as Trp

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phosphorescence probes exclusively the immediate environment of the chromophore. Additional possibility for study of alterations of protein tertiary structure in ice was provided by fluorescent probe ANS, which is feebly fluorescent in water, but its spectrum, is blue– shifted and its intensity is dramatically increased in nonpolar solvents or when it binds to nonpolar sites of protein macromolecules [85]. Strong binding of ANS to molten globule states of proteins was linked to the loss of tertiary structure [86] while it was used to characterization of transient states in protein denaturation [87,88]. It was shown with copper-free azurin, as a model protein system, that in frozen solution the fluorescence of ANS was enhanced several fold and became blue-shifted relatively free ANS. It was estimated that, at -13 oC, on average at least 1.6 ANS molecules becomes immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is affected by temperature of ice as well as by conditions that influence the stability of the globular structure. Lowering the temperature from -4oC to -18oC leads to an apparent increase in number of binding sites indicating that low temperature and/or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd complex or upon addition of glycerol to the solution but it is further enhanced in the presence of the destabilizing agent NaSCN. More detailed analysis of structural alteration in protein molecules upon freezing with ANS probe reveals some general characteristics of structure perturbation for different proteins distinguished by number of specific features [89]. As subjects for examining of protein structure perturbation upon ice formation monomers (BSA and azurin, α-amylase, βgalactosidase), dimmer (alcohol dehydrgenase from horse liver (LADH) and tetramers (alcohol dehydrogenase from yeast (YADH), lactic dehydrogenase (LDH) and aldolase (ALD) were used. In addition to wild type of azurin it was applied mutated variants in which stability and internal dynamics were changed through the creation of internal cavities and the elimination of a disulfide linkage. The creation of hydrophobic ANS binding sites in azurin, wild type and mutants, in ice accounting for the spectral blue shift and intensity enhancement, is owed to partial disruption of the tertiary structure triggered by the solidification of water. As it was shown here [85] a much greater intensity enhancement observed for mutants, compared to the wild type permitted to conclude that plasticity of globular packing, increasing with less stable globular folds, was a favorable factor which contributed to the disruption of tertiary structure upon ice formation. The largest sensitivity to ice crystals was exhibited by mutant forms in which internal cavities were created and these cavities were probably hydrated. Decrease in water activity upon ice formation causes the cavities to dehydrate and collapse with consequent rearrangement of the internal peptide structure. This process may results in widespread deformation of the native fold with the creation of a large number of ANS binding pockets. Disruption of the native structure inferred from ANS binding was found to draw a parallel with the extent of irreversible denaturation by freezethawing. After repeated cycles of freeze-thawing the data on fluorescence spectra and enhanced scattering testify that extent of azurin denaturation-aggregation is correlated to the value of amount of binding sites of ANS, increasing from wild type to mutant proteins and that among the latter species it is significantly larger with the cavity-forming mutant related to the compact ones. Irreversible damage of freeze labile proteins, often resulting in enzyme

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inactivation and/or protein aggregation, is likely to have originated from partial unfolding of globular structure in ice. The compression of fluorescence spectra of ANS characteristics in solution abovementioned proteins under freezing condition permit to conclude that perturbation of the native fold in the frozen state appears to be general for most proteins and point out that oligomers tend to be more labile than monomers, presumably, because the globular fold can be further destabilized by subunit dissociation. The studies of protein unfolding equilibrium in ice and determination of the thermodynamic stability of macromolecules under this condition, which were based on the changes of Trp fluorescence between native and denaturated states of proteins, were an important step towards better understanding of protein stability in ice [90]. As a model protein system azurin was selected for this study, which is well characterized in terms of its structure [78,91] and thermodynamic stability [92,93]. Whereas a single Trp residue located within the rigid inner core of the globular structure wrapped up by a tight β-barrel motif, its exposure to the solvent requires not less than global unfolding as confirmed by the superposition of fluorescence and circular dichroism upon denaturation [93]. During study of guanidine chloride-induced denaturation of (C112S)-azurin mutant in solution and in ice at the subzero temperature it was exhibited that the stability of native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool VL in equilibrium with the sold phase. The data establish a threshold, approximately VL=1.5%, below which in ice the protein free energy level decreases progressively related to liquid state. The sharp dependence of free energy on VL is consistent with a mechanism based on adsorption of the protein to the surface. The reduction in free energy is also accomplished by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. Thus the structural perturbation of protein induced by interactions of molecules with iceaqueous interface indicates the appearance on their surface hydrophobic areas which are not inherent for native states and these rearrangements in structure can create protein forms with high aggregation proneness. For example, the aggregate formation during freezing of human growth hormone and bovine IgG was attributed to a large ice-aqueous interface area upon rapid cooling [94,95].

Disturbance of Protein Conformation Affected by Dehydration Considerable changes in protein structure can proceed from dehydration process that is assumed occur during both freeze-thawing and freeze-drying because water is an essential factor for the formation of native protein structures [96,97]. Actually water in hydrated proteins often is classified by its freezing/melting behavior as unfreezable water and freezable water. 27 (Cryo-lett) Ordinarily water that remains unfrozen at temperatures below the equilibrium bulk water freezing temperature, in presence of ice, is called ‘unfreezable’ or ‘bound’. In many events amount of unfrozen water exceeds that expected at equilibrium and calculated for simple hydration shell. Partially it can be related to the fact that the equilibrium

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state under the experimental conditions is not always achieved owing to the depression of freezing point. Besides, the existence of unfrozen water at freezing temperatures in the presence of ice or other nucleators can be attributed to at least some effects, in particular, depression of the freezing point due to small solutes, presence of membranes, macromolecules and other hydrophilic ultrastructure and viscosity effects [98]. Therefore, dehydration of such system, especially at freezing temperatures takes a long time. The most important for existence of unfrozen water fraction is water of hydration. Hydration water is referred to the population of water molecules found near macromolecular surface with physical properties which differ from those of pure water [98]. In the vicinity of a polar or dipolar region of a membrane or macromolecule, the dipolar water molecule has a nonrandom average orientation and thus a lower potential energy than it does in bulk [99,100]. This energy may be lowered by hydrogen bonding to the surface. The specific entropy of this vicinal water is less than that of bulk water, but greater than that of ice. Specific potential energy and specific entropy are strongly dependent on the distance of the water from the surface which attracts it: the first hydration shell would be more strongly attracted than the second one, and so forth. The strength of the hydration interaction also depends on the surface: its charge, its polarization and the area density of hydrogen bonds. Removal of hydration water by freezing, evaporation or mechanical expulsion gives qualitatively similar results: the energy of hydration decreases exponentially with moving away from macromolecule surface. Solutes lower the chemical potential of water in solution, and so the hydration water in a solution has its chemical potential lowered by both the solute and the presence of the hydrophilic surface. This means that a lower sub-freezing temperature is required to remove it, or equivalently that more water of hydration is retained at the given temperature [98]. The unfreezable water constitutes a hydration range of up to ~0.3-0.4 h (h is defined as gram of water per gram of protein, mass ratio) and it remains mobile down to very low temperatures and does not crystallize even over long periods of time, independent of the rate of cooling or heating [101].The hydration water remains amorphous at ultralow temperatures. Hydrated water above this hydration range that comprises approximately 0.4-0.8 h crystallizes on slow cooling but can be vitrified with increasing rates of cooling. Colorimetric glassÆliquid transition and crystallization behavior of this vitreous, but freezable, water fraction was shown for methmioglobin (MetMb) [102]. The investigation [103] for hydrated MetMb, methhemoglobin (MetHb) and lysozyme powders determined that freezable water fraction of between ~0.3-0.4 h and ~0.7-0.8 h vitrified completely by cooling at rates up to 1500 K/min. This vitreous but freezable water fraction started to crystallization at ~210 K to cubic ice and at ~240 K to hexagonal ice. This water fraction, as the data of differential scanning calorimetry attested, subjected on reheating at a rate of 30 K/min, a glassÆliquid transition with an onset temperature of between ~164 and 174K, but the glass transition disappeared upon crystallization of the freezable water. The calorimetric features of behavior of this water fraction are similar to those of water imbedded in the pores of synthetic hydrogel, but very different from those of glassy bulk water. The distinct from glassy bulk water properties is attributed to hydrophilic interactions and Hbonds of macromolecule segments with the freezable water fraction, which thereby becomes dynamically modified. Presently it is thought the vitreous but freezable water fraction is structurally and dynamically modified by hydrophilic and H-bonded interactions with the proteins functional groups via the unfreezable water, and conversely, it can exert a

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plasticizing effect on the proteins segments. The vitreous but freezable water fraction can be related to water of the secondary hydration shell and its calorimetric features are characteristics for structural and dynamical perturbation of water in H-bonded network by interactions with a protein surface. Although in the previous study of freezable water with hydrated MetHb [104] it has been preferred its assignment to water in pores and/or loops of protein chain because of the similarity with water in the pores of hydrogel, then authors preferred the assignment in terms of secondary hydration shell on the ground of new experimental evidences, in particular, similarity of behavior for the three proteins (MetMb, MetHb and lysozyme) with different structure, an abrupt increase in critical cooling rate at a hydration value where the secondary hydration shell is expected to be completed and the remarkable similarity of this hydration value to that of the threshold value for water on hydrated BSA [105]. The secondary hydration shell is not expected to be uniformly distributed on the protein, and evidence for clustering of the water has been reported [105]. The calorimetric features of the vitreous but freezable water fraction on the hydrated proteins cannot be due to the secondary hydration shell alone but must also involve some water of the primary hydration shell [103]. For understanding of protein stability at low temperatures it is important to know whether it is possible that during vitrification of vitreous but freezable water fraction by rapid cooling, the water molecules occupy the same positions at cryogenic temperatures as at positive (ambient) or whether clustering of the water molecules occurs during cooling. It is expected that clustering of water during cooling depends primarily on the cooling rate and the degree of hydration and that it is favored by decreasing the cooling rate and increasing the hydration [106]. The effects of dehydration to protein conformation have long been a point of controversies. Dehydration can completely and irreversibly inactivate some enzymes [107]. This inactivation occurs presumably through loss of structure and dehydration-induced changes in tertiary and quaternary structure upon rehydration that was observed, for example, for dried myosin and catalase [108,109]. Three types of behavior can be generally observed for proteins upon dehydration and rehydration. First, a protein can be resistant to conformational changes during drying and therefore retain the native conformation upon rehydration. Second, a protein may unfold during dehydration but refold to the native state upon rehydration (α-lactaglobulin and lysozyme). Third, a protein may unfold during dehydration and not regain the native conformation, resulting in irreversible conformational changes and denaturation. Thus the inherent stability of a protein to survive dehydration and subsequent rehydration must be related to its capacity to resist conformational changes during dehydration or to refold into the native structure upon rehydration. The behavior of poly-l-lysine during lyophilization has been examined as a model system, to understand more fully the effect of dehydration to structural changes in polypeptide chain [110]. In solution poly-l-lysine adopts α-helical, βsheet or unordered conformations, depending on pH and temperature [11,112]. At neutral pH the poly-l-lysine exits as an unordered polypeptide. Freeze-drying induces a transition from the unordered polypeptide to the highly ordered β-sheet. Lyophilization from pH 11.2 solution where the polypeptide adopts α-helical conformation also induces the transition to βsheet, although a small amount of α-helix is apparently still observable. The heating of a solution of poly-l-lysine with pH 12.0 results in formation of β-sheet. For sample lyophilized after this treatment the β-sheet is preserved upon dehydration. So, the preferred conformation

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in the dried state is β-sheet regardless of the initial conformation in aqueous solution. The results of the poly-l-lysine studies provide strong evidence that the spectral changes in the amide poly-l-lysine region observed upon dehydration of polypeptides are predominantly related to conformational changes and that the effect of water removal independent on a conformational change is minimal. The dehydration-induced conformational transitions observed in poly-l-lysine are likely to be due to compensation for the loss of hydrogen bond interactions with water molecules during dehydration. In solution a random coil has its peptide hydrogen bonds satisfied by water molecules. Upon dehydration, these hydrogen bonds are lost. To compensate for this loss the polypeptides form intermolecular hydrogen bonds, resulting in the observed β-sheet conformation of the dried polypeptide. In absence of water the partial charges of hydrogen binding groups are less screened due to the lower dielectric environment, that increase the electrostatic attraction between dehydrated peptides. Thus in the dried state the hydrogen bond interaction energy between amide groups should be stronger than that in aqueous solution. The conformation transition observed for α-helical poly-l-lysine is also consistent with this mechanism. At low hydration levels the β-sheet conformation is energetically more favorable than the α-helix because the β-sheet requires a lower degree of solvation [13]. The β-sheet structure has a higher degree of intermolecular hydrogen bonds. Thus as the hydration shell is removed from the helical polypeptide, a transition to β-sheet can be induced. Similar changes are expected in protein behavior upon dehydration [110]. During dehydration, protein rearranges its conformation to maximize intra- and interchain hydrogen bonds to replace lost hydrogen bonds to water. Nevertheless, several different types of interactions are present in proteins, which are not in the poly-l-lysine model, including hydrophobic interactions. Thus the effects of dehydration on different proteins are more complex than the polylysine model. Significant conformational rigidity existing in some proteins leads to only a small conformational change observed after dehydration. In contrast, many proteins are unstable during lyophilization and after reconstitution lose all or part of their structure. The concept that proteins exist in numerous different conformations or conformational substates, described by an energy landscape, is now broadly accepted but the conformational dynamics is incompletely understood [114]. Protein dynamics is a function of temperature and hydration level and strongly affected by the solvent viscosity [115-118]. Thus the dynamics of protein groups depending on temperature can be important for understanding structural changes induced by freeze-thawing and freeze-drying. Generally at low temperatures three major relaxion processes that contribute to the observed dynamics are identified in the picosecond to nanosecond time range: 1) fluctuations of methyl groups; 2) fast picosecond relaxation; and 3) slow relaxion process. These processes are tightly related to hydration level of protein and its functional activity. The change in the hydration level significantly affects the dynamics and activities of protein. The enzymatic activity is essentially non measurable at hydration levels h0.5, asymptotically approaching the level of activity in dilute solution [119]. This dependence of lyzozyme activity on hydration differs from the dependence of hydrogen exchange rate on hydration, which characterizes the internal dynamics of protein molecules [120]. Detailed analysis of dynamics of lyzozyme as function of hydration and temperature in a broad pico- to nanosecond time window showed that a low temperature onset of

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hormonicity at T ~100 K is attibuted to methyl-group dynamics that is not sensitive to hydration. The increase in hydration seems first increase the fast relaxation process and then activate the slow relaxation process at h~0.2. The slow process increases sharply with an increase of hydration to above h~0.2. Activation of the slow process is responsible for the dynamical transition at T~200 K. The dependence of slow process on hydration correlated with the dependence of enzymatic activity of lysozyme on the hydration, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lyzozyme. Thus fast relaxations in hydration shell control [114] fast fluctuations in proteins. These fluctuations called solvent-slaved are similar to the α-fluctuations in glasses. They are absent in solid environment. Hydration-shell-coupled fluctuations are similar to the β-relaxation in glasses. They can be reduced or absent in dehydrated proteins, and occur in hydrated proteins even if embedded in a solid phase. They can be responsible for internal processes such as the migration of ligands within protein. The existence of two functionally important fluctuations in proteins, one slaved to bulk motions and the other coupled to hydration-sell fluctuations implies that the environment can control protein functions through different way and that no real protein transition occurs at near 200 K. Whereas hydration water of protein does not crystallize at low temperature the issue concerned nature of supercooling and its effect on protein function remains open. Conformational fluctuations are “frozen” at low temperatures which may be related to “freezing” of hydration water. Protein water system is a network of hydrogen bonds. This network has no translation symmetry and may be split into clusters with stronger internal coupling. The clusters involve a formation and breaking of hydrogen bonds which is predominantly a cooperative phenomenon. Thus the cooperativity of the network provides the coupling mechanism between conformational motions and water fluctuations. The clusters sizes decrease with decreasing hydration and the glass transition shifts in parallel to lowering temperatures, implying that small clusters are less stable than the larger ones. With a glance to the concept of existence of protein substates each protein is “frozen” in a particular substate below 200K. This distribution may also reflect the distribution of hydrogen bonds that includes a multiplicity of water states on the protein surface. The behavior of unfreezable hydrated water at low temperature can be observed at the example of protein, which is hydrated not more than 0.3 h because in a hydrated protein a fraction of water of up to ~0.3 – 0.4 h remains mobile down the to the very low temperatures and does not crystallize even during long periods, regardless of the rate of cooling or heating [121], while hydrated water above this hydration range crystallizes on slow cooling. The DSC study of hydrated MetHb, MetMb, and lysozyme powders, which have been hydrated only up to 0.3 h, (but less), at which water does not crystallize, and the complications due to ice formation on cooling are avoided, shows that their heat capacity slowly increases with increasing temperature without showing an abrupt increase characteristic of the glassÆliquid transition [122]. Annealing, which also referred to as physical aging, of the hydrated proteins is accompanied by appearance in DSC scans an endothermic region, similar to an overshoot, immediately above the annealing temperature. This annealing effect appears to occur at all temperatures between ~150 and 300 K. An increase in the annealing time at a fixed temperature leads to an increase in peak surface that attributed to the presence of a large number of local structures in which macromolecular segments diffuse at different time scales over a broad range. The lowest time scale corresponds to the >N-H and –O-H group motions which become kinetically unfrozen at ~150-170 K on heating at a rate of 30 K/min and which

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have a relaxation time 5-10 s in this temperature range. The annealing effects confirm that the individual glass transition of the relaxing local regions is spread over a temperature range up to denaturation temperature region of the proteins [122]. The possibility of coupling of internal motions of the protein with the adjacent solvent is confirmed by many works [106,123,124]. The two types of changes in solvent distribution can lead to changes in H-bond interactions of water and ice with the protein surface. According to the insights into structure and dynamics of water around macromolecules some water molecules are located at the periphery of proteins and provide the hydration shell, and the others are internal water, which fills cavities in a protein and diminishes local changecharge interactions. Molecules of water which enter in protein cavities are very mobile and they exchange easily with bulk water. Calorimetric studies of the melting patterns of ice in hydrated metMb powders at 0.43 and 0.58 h provide evidence for recrystallization of ice at subzero temperatures and subsequent clustering of water on heating to the ambient temperature. Affect of water dynamics observed in protein with different hydration level (range of 0.07-0.4 h) is not expected to be large nevertheless subtle changes seem to be possible and can be investigated [119]. Summarizing the data on protein macromolecular structural dynamics and water phase behavior at low temperatures as well as the supposed alterations in water-protein interactions one can predict the appearance of protein macromolecules with conformations which are essentially different from native ones. These changes can evolve aggregation processes that are really confirmed by many studies [125,126].

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Stabilizing Effects of Cryopotectant Agents on Proteins The destructive alterations in cells and proteins in solutions induced due to changes of environmental physical and chemical parameters that accompany the freeze-thawing or freeze-drying can be largely prevented by cryoprotective agents. Among cryoprotectants different chemical compounds are encounted including small carbohydrates (trehalose, sucrose, etc); polyols (glycerol, sorbitol, mannitol); amino acids (glycine, proline); DMSO; polymers (polyethylene glycol, polyvinilpirolidone, dextran, hydroxyethyle starch). These compounds are compatible and can generally stabilize macromolecular structure (proteins, membrane) under variety of stress conditions. Nevertheless not all of them are equally effective in respect to different types of proteins and cells upon freeze-thawing and differently withstand against stress factors of various origins. Some compounds in particularly, DMSO; PEG, ethylene glycol, which have marked cryoprotective effects, are able to exert toxic effect on cells and macromolecules at elevated temperatures [126-131]. The mechanisms of macromolecules stabilization by cryoprotectants are not completely understood but apparently certain general principles of water - solute –protein interactions should be underlaying the stabilizing effects of structurally different chemical compounds. A single universal mechanism of protein stabilization in osmolyte solution against stress impacts was proposed by Timasheff, Arakawa and their colleagues [131-134]. Among osmolytes, substances which are synthesized by living organisms under stress conditions, a group of compounds commonly used as cryoprotective agents can be separated. Further

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Carpenter and Crowe [135] expanded this mechanism towards cosolvent-induced stabilization of protein during freeze-thawing. According to this conceptualization the stabilizing effect of cosolvents based on the balance of preferential binding and preferential exclusion as consisted with that in a triple system of “water–macromolecule–solute” the solute (osmolyte, cryoprotectant) proves to be preferentially excluded from the immediate contact area around the protein molecule. Actually the effect of solute on the stability of the protein derives from the balance between destabilizing effect of solute binding and the stabilization resulting from solute exclusion from the protein. The general property of solutes, which appears as exclusion from protein surface was termed the osmophobic effect [136-138]. The main explanation of macromolecules stabilization under the effect of cosolvent compounds in solution is based on thermodynamic theory. The presence of cosolvents in protein solution creates a thermodynamic unfavorable situation since the chemical potential of both protein and cosolvent are increased. The thermodynamically unfavorable effect arises from decreasing in entropy of the entire system. As a result the native structures of monomers and oligomer forms of proteins are stabilized as processes of both denaturation and dissociation would lead to increasing in the area surface of contact between the protein and cosolvent and increment in entropy unfavorable changes in the system. If one assumes that protein was to unfold exposing more surface area, which presumably would preferentially exclude more solute, the already thermodynamically unfavorable situation would become even more unfavorable. As a result the protein does not unfold and stabilization of its native macromolecule conformation can be observed. When considering the parameters of interaction in the triple system “water (1) – macromolecule (3) – cosolvent (3)”, the preferential interactions of protein and cosolvent are described with the close approximation by the parameter (δm3/ δm2)μ1,μ3 where μ1 and m1 are chemical potential and molal concentration of component 1 (water) [139]. The positive value of preferential interaction parameter indicates an excess of component 3 in the vicinity of protein in respect to the bulk concentration. A negative value for this parameter indicates a deficiency in component 3 nearby the protein molecule and, consequently, component 1 (water) is in excess near the protein surface. The preferential interaction parameter is related to the change in the free energy of the system induced by cosolvent [131]. The compounds excluded from protein surface and possessing by the positive value of interaction parameter, increase the chemical potential of the protein, rendering the system more thermodynamic unfavorable. In the contrary, the solute that preferentially binds to proteins decreases its chemical potential and free energy. At the ambient temperature in water the native form of protein has a lower free energy than the denatured one and adding preferentially excluded cosolvent will accompany the greater exclusion for denatured state than for the native form since the former has a larger surface area of the contact with cosolvent. This means that in a greater degree the increase in the chemical potential in presence of cosolvent is observed for the denatured protein. Consequently, the increase in free energy difference between the denatured and native protein forms under the excluded cosolvent effect will stabilize the native state [131,140,141]. Preferentially bound cosolvent will bind more to the denatured protein than to the native one and it is obvious that the preferentially binding of cosolvent enhances with the increase in its concentration. In the presence of such cosolvent the free energy difference between the native and denatured states of a protein becomes smaller and then it even can reverse at a certain cosolvent concentration. In this case the native state is characterized by a higher free

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energy than the denatured state and as a result the denatured form becomes more thermodynamically favorable in the presence of preferentially binding cosolvent [131, 140142]. The same principles are applied to explain the cosolvent effect for stabilization of oligomer forms of proteins or for the degree of assembly of protein oligomers and polymers (144-146). The preferentially excluded cosolvent tends to induce polymerization since the formation of contact between constituent monomers serves to reduce the surface area of the protein exposed to the solvent. Thus polymerization reduces the thermodynamically unfavorable effect of preferentially excluded cosolvent since the increase in the chemical potential in the presence of the cosolvent is less for polymers than for constituent monomers. Conversely preferential binding of cosolvent induces depolymerization since that increase the area surface of the contact between protein and cosolvent and much cosolvent is bound. Comparing effects of exclusion during denaturation and polymerization it should mark the principal difference between these two cases [146]. During polymerization the protein conformation subjects to insignificant alterations and chemical nature of the protein surface does not change much as a result. During denaturation the conformation of the protein subjects to essential alterations and, in addition, the surface properties of protein also alters significantly. The changes in the chemical nature of the protein surface upon denaturation will enhance exclusion of stabilizing compounds. Preferential exclusion of cosolvent from macromolecule surface is not related to a certain single physic or chemical characteristic. Nevertheless among them it is possible to discriminate some groups which combined by common property that can explain mechanism of compound exclusion. For example, almost all preferentially excluded compounds except PEG, MPD, DMSO, ethanol and ethylene glycol, have strong cohesive force on water and are essentially polar. The cohesive forces of cosolvents are reflected in increasing in surface tension of water in presence of cosolvent [133,147-149]. The exclusion of these compounds is due to their surface tension increment in water. With application of the Gibbs phase isotherm the strong correlation between the value of preferential interaction parameter and the value of surface tension was exhibited. The excluded cosolvent increases in surface tension of water due to its effects on the organization of water. Such cosolvents would be excluded from the only place where it is possible, in particular air–water interface as well as from the surface of a protein. Cosolvent would tend to be included into the bulk solvent leaving water behind and become excluded from the protein surface. The other physical property, which explains the exclusion from protein macromolecule can be related to the steric hindrance mechanism proposed by Kauzmann and introduced by [150]. This mechanism is valid for such substances as PEG, MPD, DMSO, ethanol and ethylene glycol, which possess both polar and nonpolar properties and actually decrease the surface tension of water, probably because they tend to align in interface [151]. The steric exclusion of these compounds based on the magnitude of their hydrodynamic radii, for example PEG, which is much greater than that of water. This is to result in an excess of water between the protein and the hydrodynamic radius of PEG and consequently in deficiency of PEG in vicinity of the protein relative to the bulk solution [152]. This mechanism is acceptable for many cosolvents that has a larger hydrodynamic radii than water. The exclusion of MPD was shown to occur trough repulsion of MPD molecules from the charges on the protein molecule [153] due to the decrease in the dielectric constant of the medium in response to addition of the cosolvent with concomitant increase in the unfavorable

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electrostatic free energy of the charges [153]. Through the same mechanism the ethanol could operate and become excluded from a protein [151]. Finally the preferential exclusion of DMSO may occur due to its strong, nearly stoichiometric, binding to water molecules [159]. Due to its high affinity to water and much less affinity to proteins DMSO tends to remain preferentially in the bulk water. As molecule structures of several cryoprotectant agents (cosolvents) possesse significant nonpolar areas, these compounds can interact with hydrophobic surface of protein. These interactions may underlay the toxic effects such compounds as PEG, MPD, DMSO, ethanol and ethylene glycol. The hydrophobic interactions become predominated with elevation of temperature and increasing in concentration of these cosolvents. This property can be responsible for the cosolvents effects as denaturants at a higher temperature. As a matter of fact higher temperatures increase the thermal energy and make the structuring of water into clathrates near the nonpolar side residues of protein even more unfavorable. Therefore the hydrophobic interactions between cosolvent and macromolecules are enhanced under these conditions. Lower temperatures favor the structuring of water and thus decrease the hydrophobic interactions [154]. Nevertheless, when the concentration of the cosolvent elevates up to a certain critic value these interactions can predominate even at a lower temperature (below the ambient temperature). Thus due to their dual hydrophilic and hydrophobic character of interactions the molecules like DMSO and PEG can both stabilize and destabilize a protein depending on the temperature, the cosolvent concentration and the relative hydrophobicity of a protein, as well as presence of other cosolvents [155,156] and as a result be bound or excluded from the protein. Thus, on one hand DMSO and other nonpolar cryoprotectants may prevent ice formation and the concomitant freeze-thawing damage at low temperature, but on the other hand at high concentrations the same cryoprotectants can be lethal to nonfrozen cells or lead to irreversible inhibition of protein activity. In order to understand better the mechanisms of protein stabilization under the conditions of freeze-thawing it is important to realize to which extent the conception of protein stabilization against different stresses in nonfrozen solutions based on preferential exclusion of cosolvent can be expanded to the situation in the frozen state. Actually during freezethawing when protein and cosolvent remain in the liquid phase as ice is formed the same types of interactions between the cosolvent and protein are to occur during freeze-thawing as in aqueous solution. Therefore during freeze-thawing the cosolvent would also stabilize the native state of protein and decrease irreversible interaction of proteins by the same mechanism, which describes the behavior of protein in cosolvent solution at positive temperature. Over 25 preferentially excluded cosolvents examined in [135] protected lactate dehydrogenase during freeze-thawing while preferentially bound cosolvents enhanced its inactivation. It is well known that extreme dehydration often irreversibly disturbs protein structures, which eventually leads to denaturation and often to aggregation. For example, phosphofructokinase (PFK) which is a tetrameric enzyme dissociates into its component parts during drying and denatures irreversibly [157-159]. This enzyme loses all its activity when it is only partially dehydrated. But when a stabilizing solute (proline, for example) was added to the protein solution the enzyme was dehydrated to much lower water contents. Nevertheless the preferential exclusion mechanism cannot operate when all the bulk water is removed. As a result, at the lowest water contents the PFK activity declines sharply. When PFK was dried in the presence of trehalose the enzyme activity was completely preserved, even at the lowest

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water content. Many efficient cryoprotectants such as glycerol, proline, and trimethylamineoxide (TMAO), all of which are also preferentially excluded from the protein surface, have no stabilizing properties during extreme dehydration. The only solutes that do have such properties are disaccharides. To explain the disaccharides stabilizing action it was assumed that during dehydration the solute concentration must rise to the point where the solute penetrates into the hydration shell of the protein. Using infrared spectroscopy, it was shown that when trehalose was used as a stabilizing agent for drying a protein the bands assigned to OH deformations in dry trehalose were altered in ways that mimic addition of water to the trehalose. Simultaneously, dehydration-induced shifts in the amide and carboxylate bands in the protein could be partially or completely reversed in the presence of trehalose. These results suggest that there is an extensive hydrogen binding between -OH groups on the trehalose and polar residues in the protein that preserve the conformational state of the dry protein similar to that seen prior to dehydration. Although the conception of preferential solute exclusion does not include the impact of mobility change on protein stability and does not account for the protein degradation at the ice-aqueous interface many aspects of macromolecule stabilization can obtain sufficient explanations. Understanding of nature of stresses affecting proteins during freeze-thawing or freeze-drying permit to approach to conscious choice of stabilizing compounds. As an example, for stabilizing protein lactate dehydrogenase [160] during freeze-drying the maximum retention of functional activity was provide by combination of PEG as cryoprotectant and disaccharide as lyoprotectant.

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Aggregation Frozen-Thawed and Frozen-Dried Proteins [A4]The different stresses affect the protein structure stability yielding partially unfolded states with a high amount of extended chains. The consequences of structural perturbations in macromolecules often result in formation of protein aggregates, which can be both soluble and insoluble. After freeze-thawing the emergence of insoluble aggregates was observed for phosphofructokinase, glutamate dehydrogenase, lactate dehydrogenase [161] and human growth hormone [162]. Lyophylized human serum albumin upon dissolution formed a fraction of soluble aggregates [163]. Actually the consequences of protein structure perturbations during freeze-thawing and freeze-drying are dependent on many factors of protocol proceeding. Protein structures, which form during cooling, can have a strong tendency to aggregate at moderate temperatures [164]. The cold induced states of myoglobin were exhibited to be only partially unfolded and the rigid core of the G- and H-helices was maintained [164,165]. Nevertheless, the states induced by cold unfolding were not completely reversible at ambient conditions, representing a heterogeneous ensemble of refolded and unfolded species. The moderate temperatures proved to be sufficient to induce a further conformational changes inunfolded species leading to aggregation. Actually the cold unfolded state, which was similar to pressure induced state, had a high content of the extended chain (50-60%) and easily aggregated. In contrast, the heat unfolding readily led to aggregation whereby almost 30% of helix was transformed into intermolecular β-sheet, whereas the almost of the extended chain remains unchanged [164].

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The difference of structural organization of the aggregates induced by freeze-thawing in respect to thermal stress induced ones were shown for huminazed monoclonal IgG1 [166]. The structural state was characterized by spectroscopic techniques, dynamic ligh scattering, SDS-PAGE and other methods. The data obtained allow concluding that thermal stressing of IgG1 led to the formation of relatively small aggregates, which were in the size range bellow 30 nm. SDS-PAGE revealed that these aggregates were partially covalently linked via disulfide bridges and made up of conformationally perturbed monomers. Aggregation after freeze-thawing manifested itself primarily in particle formation in the micrometer range. These aggregates were noncovalently linked and composed of native-like monomers. The lack of significant structural changes observed for cold-induced aggregates [166] could be in agreement with the reversibility of structural changes, if any, induced by freeze-thawing. However surface-induced structural alterations in the frozen state might function as trigger for the observed particle formation. Summarizing the data demonstrated by combining set of analytical techniques one can see that heating and freeze-thawing of monoclonal IgG1 antibody result in the formation of aggregates, which differ considerably in their size and structure. Actually the protein aggregation during freeze-thawing is attributed to partial unfolding of protein molecule [169] caused by the perturbations of environmental conditions arising during the proceedings. Among parameters of solution that can affect protein structure and contribute to aggregation are pH and ionic strength, which modulate both the conformational and colloidal stability of protein [170] and define the protein response to other physical stresses accompanying the freeze-thawing. In addition, the warming and cooling rates applied during freeze-thawing of a sample, which determine the “concentration solution” effects [171] and duration of exposure of the protein under these potential stresses, can affect the degree and rate of aggregation. It was shown that aggregation of the antibodies during freezethawing increased with decreasing pH, which correlated well with Tm values [169]. Aggregation was the most prevalent at pH 3 - 4 with potential mechanisms involving both the formation of aggregation-prone conformational states as well as adsorption to and denaturation at various interfaces. Besides high concentrations of KCl contributed to aggregation at pH 3 that was probably due to shielding of charged residues on the protein, resulting in a reduction of charge-charge repulsion and making intermolecular interactions more favorable. The presence of the salt is likely to lower the glass transition temperature of non-ice phase, providing additional time for protein aggregation before glass formation. In contrast, at pH 4 the salt mostly reduced aggregation. Actually, it is well known that salts can affect the conformational stability of proteins via preferential interactions and thus serve as stabilizers or destabilizers. This mechanism can be involved in different protein behavior at pH 3 and pH 4 at the same salt concentration. Another parameter affecting the protein aggregate formation during freeze-thawing can be a material of container that probably related to some extent to the nature of adsorption interface. Actually material, geometry and volume of the container can affect protein damage during freeze-thawing by modulated the effect of adsorption of protein molecule at the liquid-container interface and by alteration of cooling and warming rates. The importance of container materials for protein aggregation during freeze-thawing was exhibited for IgG2 [169]. Samples stressed in plastic or glass container material comprised low amounts of aggregates. Storage in Teflon or commercial freezing containers, however, led to significantly larger level of aggregate formation.

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Generally, the investigation of protein aggregates induced by various stresses including freeze-thawing and freeze-drying is difficult for some reasons [172]. Aggregates are generally structured but not sufficiently ordered and homogeneous to produce high-quality crystals for X-ray structure determination, besides the solution-phase NMR is untenable for aggregate study due to the protein transformation into insoluble aggregates at the high concentrations required. Tentatively, one can say that hydrophobic collapse is one of the most important driving forces for aggregate formation and the extent of organization can vary from some aggregates being highly crystalline to others which are more accurately classified as micellar assemblies. There may be a considerable heterogeneity among aggregates in packing of subunits into hierarchical structures. Some protein aggregates retain structures very similar to the native fold [163]., others contain folds completely different from the native protein [173], and sometimes it is possible to observe complete rearrangement into folds not normally seen in globular proteins (e.g, β-helix). In general the pathways by which polypeptides misfold and aggregate and the stability of the aggregates relative to the natively folded protein and to any misfolded intermediates, are of considerable interest. All this concerns to the full extent the mechanisms of protein aggregation after freeze-thawing and freeze-drying. Protein aggregation pathways can be categorized as one of the two alternatives: rapid hydrophobic collapse into quasi-stable oligomeric intermediates that slowly mature to a stable aggregate, or rare conformational conversion from a natively folded protein to a partially misfolded "nucleus" followed by rapid growth. Four general, somewhat overlapping, pathways towards aggregated proteins were considered in [174]: 1) monomer-directed conversion, in which collision of two monomers induces conversion to an aggregation-prone conformation, 2) nucleated polymerization, in which a rare thermodynamically unfavorable conformer appears and nucleates further growth by monomer addition, 3) templated assembly, in which monomers deposit onto a pre-existing aggregate, and 4) nucleated conformational conversion, in which collapse of monomers into an oligomer precedes a conformational conversion to a structured aggregate. For understanding of protein aggregation mechanisms it can be useful to realize clearly the similarity and difference between oligomer formation and their stabilizaty and the process of aggregate formation. Many proteins which function as dimmers or tetramers have a potential to prevent their aggregation due to quaternary structure that greatly stabilizes proteins against the aggregation. Nevertheless dissociation of stable oligomers to monomers induced by any changes in solution conditions can be a factor initiating of aggregate formation. By the example of transthyretin [175] and insulin [176] it was shown, that slight changes in monomer conformation determined whether self-association would lead to native oligomers of the defined size or to large non-native aggregates. Some aggregates might, thus, be considered as alternative quarternary structures. Along these lines it is interesting to note that some cosolutes that are known to stabilize native folded proteins, also accelerate aggregation of unfolded polypeptides [177,178]. This is presumably due to the fact that these cosolutes increase the hydrophobic interactiona that drive both native folding and misfolding and aggregation [179]. Many solutes, which stabilize proteins through preferential exclusion, also drive self-association of the native state. Solute conditions, which lead to unfolding, will often lead to irreversible aggregation. As it was mentioned above cosolvent might inhibit aggregation by stabilizing the native state through a preferential exclusion mechanism. Soluble aggregates may be formed by mechanisms that involve either covalent or noncovalent linkage of protein monomers. In most cases protein aggregation results from

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noncovalently linked macromolecules, whose structure is altered often due to partial denaturation with exposure of hydrophobic regions that more readily associate forming protein-protein contacts. Conversely, the formation of covalently linked aggregates may or may not involve a change in protein structure. For instance, a properly folded protein that contains a noncoupled thiol may form aggregates by intermolecular thiol exchange. On the other hand, protein structural alteration may result in disulfide bond breakage, and the resulting free thiol may participate in the disulfide exchange reaction. In the case of frozendried rhuMAb, the aggregates, which occurred during storage were characterized as native protein molecules that were linked by intermolecular disulfides[180]. Thus protein aggregates observed following the storage and reconstitution of frozen-dried rhuMAb formulations consisted of antibody dimers and trimers that were covalently linked by intermolecular disulfides. The preparation of frozen-dried rhuMAb formulated without excipient resulted in an irreversible solid-state protein structural alteration. This was likely to be caused by the removal of tightly bound water from the protein surface by excessive dehydration and correlated with increased rates of formation of the protein aggregates during storage. The addition of the carbohydrate excipients sucrose or trehalose to the formulation provided a solid-state environment where complete coverage of the protein surface-accessible hydrogen binding sites was achieved. This correlated with the improved native-like solid-state protein structure and reduced protein aggregation during storage Tendency to aggregate formation induced by protein destabilization as a result of changes of solution conditions is dependent on both colloidal and conformational stability. It was shown [181] that aggregation of recombinant human granulocyte stimulating factor (rhGCSF) first involved the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate that is a common feature for perturbations induced by different stress factors. Changes in the free energy of unfolding (characteristic of protein stability) and the osmotic second virial coefficient B22, (a characteristic of colloidal stability) are important parameters of protein-protein interactions. Thus both these properties are dependent on the solution conditions and either of them could be rate-limiting for aggregation proceeding. The osmotic second virial coefficient is a measure of nonideal solution behaviors that arise from two-body interactions. B22, denoting protein-protein interactions, is related to the intermolecular separation distance, the interaction potential, and temperature. The interaction potential describes all of the interaction forces between two protein molecules, which include hard-sphere, electrostatic, van der Waals, and other short-range interactions. Positive B22 values indicate overall dominance of repulsive forces between proteins, where protein-solvent interactions are favored over protein-protein interactions [182,183]. Negative B22 values reflect attractive forces between proteins and protein-protein interactions being favored over protein-solvent interactions. Assembly of protein molecules into non-native aggregates, resulting in either ordered aggregates (e.g., amyloid fibrils related to certain human diseases) or disordered aggregates (e.g., inclusion bodies and amorphous precipitates), further can involve the formation of higher molecular weight assemblies from initial lower molecular weight species. Actually, the same intermolecular interactions that govern the formation of non-native protein aggregates are also expected to be important in protein crystallization and salting out [182-184]. But in contrast to crystallization and salting out, aggregation is a more complicated phenomenon. The intrinsic conformational stability of the protein native state plays an important role in its

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propensity to aggregate. Protein aggregation typically is accompanied by large changes in the secondary and tertiary structures of the protein,. Intermediates that are structurally expanded in respect to the native state were found to precede aggregation [185]. Perturbation of a protein native structure related to formation of an aggregation competent species is likely the first conformational change that occurs along the aggregation pathway (Fink 1998; Kendrick et al. 1998).Thus aggregation is governed by the thermodynamic stability of the native state relative to that of the aggregation competent species. Then the formation of higher molecular weight aggregates from monomers must involve assembly processes, which are mediated by molecular interactions, and hence, governed by colloidal stability. Two major contributions to interactions between colloids in aqueous solutions are Coulombic electrostatic interactions and van der Waals interactions [188]. The sum of these forces describes the net force affecting colloidal particles. Due to the structural, functional, and surface anisotropy of protein molecules, their interactions are often dominated by contributions from relatively few, high-energy intermolecular configurations rather than by the overall colloidal interactions [189]. Protein molecules approaching each other should overcome an energy barrier to come into physical contact. Increasing salt concentration decreases electrostatic repulsion and thus lowers an energy barrier and at high enough concentrations particles become unstable and coagulation occurs. Interaction energy between protein molecules thus controls the energetics of their assembly processes. For example, the net charge on rhGCSF changes from +14 to -4 as pH increases from 3.5 to 7. When a protein carries a net charge, electrostatic proteinprotein interaction is invariably repulsive. Clearly, electrostatic repulsion cannot be dominant at pH 7, where interactions are overall attractive, as evidenced by the negative B22 value. In addition to electrostatic interactions, anisotropic charge distribution could give rise to highly attractive dipole-dipole interaction configurations[190,191]. This is likely the case with rhGCSF, because the locations of the 9 positive and 13 negative residues in its crystal structure show highly asymmetric surface distribution at pH 7. Highly attractive dipole-dipole interactions are also likely to be the cause of the negative B22 value measured at pH 6.1, the isoelectric point of rhGCSF. At pH 3.5, all charged residues in the protein are positively charged, thus greatly reducing dipole moments and causing rhGCSF interactions to be repulsive at all separations and orientations. Thus non-native aggregation of a protein involves at least two processes, including conformational changes in the protein native state, and assembly of protein molecules into higher order aggregates. The energetics of these processes is controlled by conformational stability and by colloidal stability, respectively. The aggregation of rhGCSF in solution conditions, where the native state is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit, first should involve the perturbation of its native structure to form a structurally expanded transition state and then it can follow by dimerization to form an irreversible aggregate or other aggregate forms. Both colloidal and conformational stabilities are expected to be important for aggregation of other proteins. Therefore, to successfully stabilize protein against aggregation, solution conditions need to be chosen not only to stabilize the protein native conformation, but also stabilize protein against attractive intermolecular forces. Reduction of liquid phase volume during solidification upon decrease in temperature is related to a crowding phenomenon to some extent. Generally macromolecular crowding, a

Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins [192]. An obvious, but important, consequence of macromolecular crowding is the excluded volume effect due to the mutual impenetrability of the macromolecules, resulting in a reduction in the accessible space for these molecules Crowding can prevent stress-induced aggregation and misfolding of proteins by altering the folding kinetics [194] and affecting the thermodynamic stability. Mechanisms of crowding effects on protein folding remain controversial. For example, some theories predicted a modest effect on the thermodynamic stability by crowding [195 while others predicted significant stabilization (Minton 2005). Similarly, conflicting results were reported from experimental studies. It was found that macromolecular crowding could accelerate a fast-track folding process, but decrease a slow-track folding process of hen lysozyme. It was reported [196] that some proteins (dihydrofolate reductase, enolase, and green fluorescent protein) could fold spontaneously in dilute, but not in crowded, solutions. The influence of crowding conditions to protein stability could be expanded to some extent to the cases of freeze-thawing and freeze-drying. Thus, protein aggregates rising from freeze-thawing and freeze-drying can differ by size, structure, solubility, and their stability/reversibility. Mostly they represent structures formed via noncovalent interactions, although covalently linked ones are often encountered, especially among lyophilized proteins. The main promoting factor of protein aggregation induced by low temperatures and concomitant physical stresses is formation of nonnative structure of proteins which are more or less stable and readily involved in further transformations related to aggregation.

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Conclusion The freeze-thawing and freeze-drying of biological systems are complex processes aimed to preserve native structure of different biomacromolecules. Nevertheless numerous stresses rising from lowering temperature in liquid phase and crystallization with concomitant changes in environment can affect protein structure. As a result there is probability of accumulation of structural states distinct from native one. Such states can be both reversible and irreversible that depends on inherent properties of individual proteins and specific conditions of environment. Cold denaturation contributes to formation of macromolecule states with exposed to solvent hydrophobic side groups of polypeptide chain and impairment of protein structure. The stability of these forms can be promoted by changes in pH and ionic strength and leads to protein aggregation. Ice crystals formation is considered to be the main factor of protein injuries during freezing, negatively affects macromolecule conformations and contributes to exposure of hydrophobic side groups of chain to solvent resulting in rise of nonnative structures of proteins, which have strong aggregation prone tendency. Surface area of ice-aqueous interface, which is dependent on many variations in environment conditions of freezing process, is the most important factor affecting protein structure. Besides, the behavior of macromolecules and hydrated water during freezing, thawing and drying can determine structural changes in proteins due to modified dynamics of macromolecules and altered water structure at low temperatures as well as during cooling,

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warming and annealing. Dehydration of proteins can result in structure transformations and onset of nonnative states, which interact with other molecules to compensate loss of H-bonds and forms aggregate complexes. Structural transformations in proteins during freeze-thawing and freeze-drying sometimes can affect sulphonic groups and involve them in interactions between macromolecules with formation of covalently linked aggregates unlike most of aggregative forms which are formed due to noncovalent interactions and more characteristic upon freezing. Stabilization of protein structure against freeze-thawing and freeze-drying is achieved by application of cryoprotective agents, which are not restricted by a certain type of chemical compounds. The mechanisms of macromolecules stabilization by cryoprotectants are not completely understood; nevertheless the common opinion on their stabilizing effect is based on the phenomena of preferential exclusion of cosolvents from protein surface and preferential hydration of protein macromolecules. Actually, the effect of cosolutes on the protein stability derives from the balance between destabilizing effect of solute binding and the stabilization resulting from solute exclusion from the protein. As a result, native state of protein proves to be stabilized and aggregation of macromolecule is mostly avoided. Nevertheless cryoprotectants differ in their stabilization efficiency and some of them possess toxicity that depends on many environment conditions and inherent protein structure. Therefore approach to proteins protein stabilization during freeze-thawing and freeze-drying is more or less empirical and to overcome these limitations further investigations are required.

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ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 2

Molecular Chaperones and Proteases as Suppressors of Protein Aggregation in Gram-Negative Bacteria Joanna Skorko-Glonek*, Dorota Kuczynska-Wisnik, Dorota Zurawa-Janicka, Ewelina Matuszewska, Donata Figaj and Barbara Lipinska

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University of Gdansk, Department of Biochemistry, Kladki 24, 80-822 Gdansk, Poland

Abstract Protein misfolding is a detrimental and often deleterious phenomenon leading to a loss of functionality of a protein, formation of improper interactions or in many cases to aggregation. This process occurs efficiently especially under stressful conditions, e.g. exposure to high temperatures, oxidative or reducing agents, or as a result of certain mutations. Although bacteria are not endangered by numerous diseases associated with protein aggregation (unlike mammals), formation of aggregates represents a serious threat for these prokaryotic cells as well. In this review we present the current knowledge concerning the bacterial protein quality control systems. In the response to presence of misfolded proteins the specially dedicated defense systems are induced. They comprise molecular chaperones and proteases, whose function is to bind, prevent aggregation and refold polypeptides, or to degrade the irreversibly damaged proteins, respectively. In the cytoplasm of Gram-negative bacteria there are two major chaperone systems: (1) DnaK-DnaJ-GrpE and (2) GroEL-GroES. They collaborate with other chaperones: ClpB and small heat shock proteins IbpA and IbpB. The major cytoplasmic proteases are Lon, ClpAP, ClpXP, HslUV. The cytoplasmic protein quality control system is dependent on energy supply from ATP hydrolysis. The periplasm lacks the classical chaperone systems but it contains a group of proteins collectively named *

Email: [email protected]

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Joanna Skorko-Glonek, Dorota Kuczynska-Wisnik, Dorota Zurawa-Janicka et al. “folding helpers”, comprising also proteins with chaperone-like activity. They include SurA and Skp proteins engaged in folding of the outer membrane proteins. The major periplasmic protease is HtrA, responsible for degradation of misfolded proteins within the cellular envelope. It shows also a chaperone activity. Protein aggregation in bacteria represents also a serious problem in biotechnology as many heterologous proteins expressed in bacteria are deposited in form of inclusion bodies. Proper adjustment of the levels of molecular chaperones is one of the solutions.

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Introduction Gaining the proper conformation is crucial for a protein to fulfill its biological function. In principle, the amino acid sequence of a polypeptide contains all necessary information for a protein to achieve the native conformation. However, the high protein concentration in the living cell does not promote spontaneous folding and favors rather incorrect interactions. Therefore, the process of protein folding is catalyzed by a group of specially designated proteins, collectively named folding factors (Schlieker et al, 2002). This group comprises: (1) molecular chaperones, whose function is to bind unfolded polypeptides, prevent their aggregation and facilitate their folding; (2) peptidyl-prolyl isomerases (PPIases), which catalyze cis-trans isomerization of peptidyl bonds; (3) oxidoreductases (Dsb protein family), which catalyze disulfide bond formation and isomerization. Under physiological conditions, these factors are in principle sufficient to maintain the quality control of protein biogenesis. However, bacterial cells have to cope with various environmental conditions during their life cycle. These include variable temperature and pH, oxidative agents and a wide range of toxic compounds. The exposure of a cell to stressful conditions damages various cellular components, including proteins, which may lose their native conformations or be unable to fold correctly. The misfolded proteins may also appear as a result of overproduction of recombinant proteins or mutations affecting protein folding. Such proteins are usually not only inactive but may also interact with inappropriate partners and/or aggregate. To prevent these detrimental effects, cells induce sophisticated defense systems, whose role is to sequester unfolded polypeptides from other cellular components, suppress their aggregation and eventually refold the denatured proteins. The irreversibly damaged proteins are directed to degradation. These functions are predominantly carried out by molecular chaperones and proteases, respectively. Most of them belong to a group of stress proteins, collectively named heat shock proteins (HSPs) (Weibezahn et al., 2004). The cells of Gram negative bacteria are composed of two separate compartments: the cytoplasm and the cellular envelope. These two spaces differ significantly in physicochemical properties and in metabolism. Therefore the separate protein quality control systems for the cytoplasmic and extracytoplasmic proteins evolved. The cytoplasmic systems rely on ATP as the energy source, whereas the periplasmic folding factors and proteases are ATPindependent.

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The Cytoplasmic Protein Quality Control Systems The cytoplasm of Gram negative bacteria is the space where most metabolic reactions occur and is separated from the environment by the cellular envelope. Here the proteins are synthesized. The nascent polypeptides are either folded to their native conformations or directed to the extracytoplasmic locations. ATP is the main source of energy for the cytoplasmic protein quality control systems.

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Cytoplasmic Chaperones The molecular chaperones are proteins which interact with non-native proteins, prevent their nonspecific aggregation and assist folding process but do not occur in the final functional structures. Stress conditions induce the synthesis of many chaperones, which belong to the group of heat shock proteins (HSPs). Chaperones are involved in many cellular functions: they assist the de novo protein folding, assembly of oligomers or protein transport, and are the central cellular defense against protein damage induced by stress conditions (Hartl, 1999). They prevent the aggregation of misfolded protein species and repair damaged or aggregated proteins. Chaperones recognize hydrophobic amino-acid side chains exposed by non-native proteins, bind them and promote folding of proteins in the ATP-dependent manner. Chaperones are classified into families according to their molecular weight and function (Hsp40, Hsp60, Hsp70, Hsp90, Hsp100 and small Hsps). Depending on their mode of action, folder chaperones, holder chaperones and disaggregating chaperones can be distinguished (Schlieker et al., 2002). Folder chaperones (like Hsp70 and Hsp60) typically utilize ATP and play an active role in protein folding and unfolding by the cycle of substrate binding and release. Holder chaperones (e.g. sHsps) only bind to the non-native proteins. Disaggregating chaperones (e.g. ClpB - a Hsp100 family member) bind and dissolve protein aggregates into smaller particles which can be further utilized by folder chaperones. Chaperones form a functional network in which holder chaperones prevent irreversible aggregation of misfolded proteins and folder chaperones actively assist protein folding. In bacterial cells two main chaperone systems function in efficient folding during the de novo protein synthesis and prevent protein inactivation in stress conditions: the DnaK (Hsp70)/DnaJ/GrpE complex and the GroEL/GroES (Hsp60/Hsp10) chaperonin complex (Langer et al., 1992; Wonga & Houry, 2004) (Figure 1). The in vitro studies indicate that these two chaperone complexes interact with the similar subsets of proteins but at different stages of the folding.

Hsp70 System Hsp70 is one of the most highly conserved family of the heat shock proteins. The DnaK protein from E. coli was the first Hsp70 protein to be discovered. Specific members of this family and their functional counterparts were then found in all species examined so far, from bacteria to higher eukaryota. Hsp70 is one of the most abundantly induced proteins under various stress conditions (Mogk et al., 1999; Bukau et al., 2006). The Hsp70 family members participate in plethora of folding processes in the cell, including the de novo protein folding,

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transport of proteins across membranes, control of protein activity, refolding of aggregated and degradation of thermally denatured proteins.

Figure 1. Hsps protect the cytosolic unfolded proteins against aggregation.

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Extracellular stress factors affect protein folding and cause protein misfolding, inactivation and aggregation. Molecular chaperones recognize hydrophobic amino-acids side chains exposed by non-native proteins, interact with them preventing nonspecific aggregation and assist the folding process. In bacterial cells two chaperone systems function in preventing protein inactivation in stress conditions, the DnaK (Hsp70)/DnaJ/GrpE complex and the GroEL/GroES (Hsp60/Hsp10) chaperonin complex. Small heat shock proteins (sHsps) function as the ATP-independent molecular chaperones. sHsp bind unfolded proteins, protect them from irreversible aggregation and facilitate their refolding by the ATP-dependent chaperones including Hsp70/Hsp40 and Hsp100. Insufficient amount of molecular chaperones leads to accumulation of unfolded proteins and protein aggregates.

The protein folding activity of Hsp70 is modulated by collaboration with chaperones of the Hsp40 (DnaJ) family and nucleotide exchange factors (GrpE). Many organisms possess multiple members of Hsp70 family. E.coli encodes three Hsp70 genes (DnaK, HscA and HscC) and six Hsp40 family members (DnaJ, CbpA, DjlA, HscB, DjlB and DjlC) (Genevaux et al., 2007). The DnaK-DnaJ-GrpE is the main chaperone system and the only one induced by heat-shock. DnaK can also productively interact with CbpA and DjlA co-chaperones. CbpA (curved DNA binding protein A) is a 33-kDa protein that was shown to act as a multicopy suppressor for dnaJ mutations. DjlA is a 30-kDa type III membrane protein required, together with DnaK, for induction of the colonic acid capsule. Both, CbpA and DjlA was shown to be able to replace DnaJ in vitro as a cochaperone for DnaK and can partially substitute for DnaJ in the DnaK-mediated protein disaggregation process in vivo (Gur et al., 2004). HscA (heat shock cognate 66-kDa; Hsc66) is a constitutively expressed Hsp70 class molecular chaperone that functions in the biosynthesis of iron-sulfur cluster-containing proteins. HscA posses a low intrinsic ATPase activity which is stimulated by HscB. HscA interacts selectively with conserved sequence present in the [FeS]-scaffold protein IscU. Expression of hscA is induced by cold shock and an hscA mutant has no detectable growth defects (Silberg et al., 2004).

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HscC, with a molecular mass of 62 kDa, is the smallest among the members of the Hsp70 family in E. coli and belongs to the most distant Hsp70 relatives of DnaK (approximately 30% amino acid identity with DnaK and HscA). HscC displays significant ATPase activity stimulated by DjlB and DjlC co-chaperones. ATPase cycle of HscC and HscA does not rely on GrpE. HscC is not able to complement the growth defect phenotype of ΔdnaK52 mutant. Differences in substrate specificity of HscC as compared with DnaK is also observed (Kluck et al., 2004). The Hsp70 molecule consists of two domains: the N-terminal ATPase (NBD – nucleotide binding domain) and the C-terminal peptide - binding domain (PBD). The major part of Cterminal region of PDB forms a helical lid. Binding and release of a substrate is controlled by ATP hydrolysis in cooperation with Hsp40 and nucleotide exchange factors. These processes are achieved by the aid of allosteric interactions between the NBD and PBD (Vogel et al., 2006). Binding of ATP to the NBD domain induces conformational changes in the peptidebinding domain which opens the helical lid to allow substrate binding. Hydrolysis of ATP, induced by the co-chaperone Hsp40, stabilizes the Hsp70-substrate complex by leading to closure of the lid (Mayer et al., 2000; Young, 2010). Binding of GrpE, the nucleotide exchange factor, is critical to Hsp70 function. GrpE binds to the ATPase domain of Hsp70, promotes the ADP-ATP exchange and triggers the substrate release. The Hsp70 chaperones interact with almost all unfolded proteins. The binding motif recognized by DnaK consists of a core of 5-7 amino acids enriched with hydrophobic residues, flanked on both sides by positively charged regions (Mayer et al., 2000). Such domain is abundant in protein sequences, and occurs every 50-100 residues. The association of Hsp70/Hsp40 chaperones with the hydrophobic patches of substrate molecules prevents aggregation of non-native proteins and reduces the improper intermolecular interactions. The DnaK system is the central holder chaperone that prevents aggregation of a wide variety of heat shock denatured proteins in E.coli. The Hsp70 chaperone system possesses also folding activity and assists non-native proteins to fold to their native state (Kurt et al., 2006). The fast folding proteins released from Hsp70 will fold spontaneously to the native state. However, certain proteins require either rebinding to Hsp70, or interaction with a chaperonin for the complete folding (Genevaux et al., 2007; Hartl & Hayer-Hartl, 2009; Young, 2010). The Hsp70s participate also in solubilization and refolding of aggregated proteins. Protein aggregates can be solubilized by Hsp70 in cooperation with chaperones of the Hsp100 family (Hsp104/ClpB) and subsequently refolded into the native state. Hsp60 System The Hsp60 proteins (also known as the chaperonins) are found in almost all organisms. They form large cylindrical complexes required for the folding of other proteins in an ATPdependent manner (Horwich et al., 2007). Chaperonins are involved in successful folding, sorting, transmembrane transport, and assembly of oligomeric polypeptide complexes (Hartl & Hayer-Hartl, 2009; Tartaglia et al., 2010). Under stress conditions they also play role in stabilizing polypetides and preventing their irreversible aggregation. In E.coli the member of Hsp60 family is GroEL. GroEL and its co-chaperonin GroES are coexpressed from a common operon (groE) and both groE gene products are essential for bacterial growth at all temperature conditions (Fayet et al., 1989). All chaperonins form cylindrical, double-ringed complexes. The bacterial chaperonins have two seven-membered rings and are assisted by the single-ring co-chaperonins (Hsp10).

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The mechanism of action of the E.coli GroEL/ES system has been well defined. GroEL subunit of ~57 kDa consist of three domains: equatorial ATPase domain, intermediate hinge domain and apical substrate binding domain. GroES forms heptameric ring of ~10 kDa subunits. Exposed hydrophobic regions of a non-native protein are recognized by the hydrophobic residues located on the apical domain of the central cavity of GroEL. Substrate polypeptides are captured by binding to three or four out of the seven sites on one open ring of GroEL. Binding of ATP and GroES to one ring induces conformational changes that lead to formation of the hydrophilic cavity in which a substrate protein is trapped. ATP hydrolysis in the GroES-bound ring and binding of ATP to the second GroEL ring results in GroES dissociation. The substrate protein leaves the cage and either folds rapidly or rebinds to GroEL for the next cycle (Xu et al., 1997; Horst et al., 2005; Tyagi et al., 2009). HtpG (Hsp90) Hsp90 is a highly conserved, multidomain protein present in the cytoplasm of eubacteria. HtpG, the E. coli homolog of Hsp90, shares a high degree of sequence similarity with eukaryotic Hsp90. All of the Hsp90 proteins form dimers of the elongated subunits, comprised of three major domains: the N-terminal ATP binding domain with affinity binding site for geldanamycin (anti-tumor drug which inhibits the Hsp90 activity); the charged middle domain that functions in substrate protein binding and in regulation of the ATPase activity, and the C-terminal dimerization domain, which possesses the ATP-independent chaperone activity (Panaretou et al., 1998; Young et al., 2001; Shiau et al., 2006). Substrate proteins are transiently enclosed by the chaperone between the two subunits. In vitro, at high concentrations, Hsp90 can act as a holder chaperone towards a wide range of substrates. Hsp90 prevents aggregation of non-native proteins that show a high degree of secondary structure. It maintains them in the folding-competent state and the subsequent action of other chaperones makes their complete folding possible (Krukenberg et al., 2008). The role of Hsp90 in bacterial cells is still not well understood. The deletion or insertion of the htpG cause a decrease in the E. coli growth rate at elevated temperatures but does not affect the protein aggregation (Bardwell & Craig, 1988). In eukaryotic organisms Hsp90 is essential for viability at all temperatures, however no increase in aggregation of proteins synthesized in Hsp90-deficient cells is observed. It is suggested that the majority of proteins do not require the assistance of Hsp90. Nevertheless, Hsp90 may play an important role in the proper folding of a selected group of proteins and under stress conditions it enhances the rate at which a heat-damaged proteins are reactivated. sHsps The family of the small heat shock proteins (sHsps) is one of the most diverse groups of stress proteins. The large majority of bacteria contain only a small number of sHsps. E. coli encodes two sHsps homologs - IbpA and IbpB (Allen et al., 1992), Salmonella enterica serovar Typhimurium expresses three sHsps - IbpA, IbpB and AgsA (Tomoyasu et al., 2003), a marine bacterium Vibrio harveyi contains a single sHsp (Klein et al., 2001). In genomes of Hemophilus influenzae or Helicobacter pylori sHsps homologues have not been found (Laksanalamai & Robb, 2004). To date the only bacteria known to encode multiple sHsps are the rhizobia (Munchbach et al., 1999). The homology between sHsps isolated from different sources is lower than in the other stress protein families (Caspers et al. 1995). Typical features of sHsps are: low monomeric molecular mass (15-40 kDa), the presence of a

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conserved region of ~90 amino acid residues, termed α-crystallin domain and the formation of large oligomeric complexes (Caspers et al. 1995; MacRae, 2000; Fu et al. 2006). Under thermal stress conditions sHsps are the most strongly induced heat shock proteins (Mogk et al., 1999). The level of sHsps also increases in the response to oxidative (Ma et al., 2006) and osmotic stress (Ventura et al., 2007), heavy metal stress (Arrigo et al., 2005; Perez et al., 2007) or cold shock (Wu et al., 2007). Small heat shock proteins function as molecular chaperones, protecting other proteins from irreversible denaturation and aggregation during physiological stresses (Jacob et al., 1993; Thomas and Baneyx 1998; Kuczynska-Wisnik et al., 2002). Numerous in vitro experiments suggest that sHsps activity does not require ATP hydrolysis; therefore sHsps become particularly important under stress conditions, when the level of ATP is extremely low. Many members of the sHsp family are inactive or only partially active under physiological conditions. In the activation process of sHsps the subunit exchange plays an essential role. The sHsps family members form continuum of oligomeric structures, from ordered symmetric assemblies to polydisperse oligomers with variable numbers of subunits (Kim et al., 1998; van Montfort et al., 2001; Horwitz et al., 2004). The high oligomeric structures serve most probably as the inactive storage forms of sHsps under physiological conditions. Dissociation of multimers can lead to the exposure of hydrophobic sites – the putative substrate binding sites, which are buried in sHsp oligomers at normal temperatures (Shearstone & Baneyx, 1998; Fu & Chang., 2004). Studies on the eukaryotic sHsps suggest that sHsps form stable complexes with their substrates and maintain them in the refoldingcompetent state until they can be disaggregated and refolded by other chaperones (Ehrnsperger et al., 1997). Binding capacity of sHsps is very high – one denatured protein molecule per subunit or dimer of sHsps oligomer. sHsps interact with a wide range of substrate proteins without sequence or molecular mass range specificity, provided that these proteins are in the unstable molten globule state (Carver et al., 2002; Kumar et al., 2005). Upon stress termination, substrates trapped by sHsps are released from the complex and refolded by the Hsp70/Hsp40 ATP-dependent chaperones (Veinger et al., 1998; Matuszewska et al., 2005). In E. coli two sHsps, IbpA and IbpB, that share 50% amino acid homology, cooperate in stabilization of denatured substrates in a disaggregation-competent state but their roles in stabilization of protein aggregates are quite distinct. The presence of both IbpA and IbpB during substrate thermal inactivation stabilizes thermally aggregated proteins in a disaggregation competent state and facilitate their refolding by the DnaK/DnaJ/GrpE-ClpB system (Matuszewska et al., 2005). However, the presence of IbpA alone reduces the ability of substrates to refold. At high temperature IbpB associated with IbpA or released IbpA mediate the inhibitory effect as well (Ratajczak et al, 2009; Ratajczak et al., 2010). The in vitro experiments indicate that the DnaK-DnaJ-GrpE system is able to efficiently refold substrates released from the soluble sHsp-substrate complexes. The refolding of proteins sequestered with sHsp in large insoluble aggregates requires, apart from Hsp70, an additional protein belonging to the AAA+ superfamily - ClpB (Matuszewska et al., 2005; Mogk et al., 2003; Haslbeck et al., 2005). Hsp100 The Hsp100 chaperones are members of the AAA+ superfamily (ATPase associated with variety of cellular activities). The AAA+ proteins are involved in a range of cellular processes

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including protein refolding and degradation, membrane fusion, vesicle trafficking and fusion, and regulation of stress response (Neuwald et al., 1999; Ogura & Wilkinson, 2001). They are found in all kingdoms of living organisms and act as energy-dependent unfoldases of macromolecules (Erzberger & Berger., 2006; Snider et al., 2008). Hsps100 usually form ringshaped oligomers with a central channel (Mogk et al., 2008; Hanson & Whiteheart, 2005). The common feature of this superfamily is the presence of the highly conserved region of 200-250 amino acids, called AAA+ domain, which participates in ATP binding and hydrolysis. The AAA+ superfamily is divided into two classes according to the number of the AAA domains. Class I contains proteins with two AAA domains (ClpB, ClpA, ClpC). Class II proteins (ClpX, HslU) contain only one AAA domain (Figure 2a) (Doyle & Wickner, 2008; Mogk et al., 2008). In both classes the ATPase domains form a channel for translocation of unfolded polypeptide chain. The majority of members of Clp/Hsp100 family act in proteolysis and will be described in the paragraph “Proteolysis in the bacterial cytoplasm”. ClpB does not associate with peptidases. It is involved in disaggregation of insoluble protein aggregates and is important for the thermotolerance in bacteria (Sanchez & Lindquist, 1990; Sanchez et al., 1992; Parsell et al., 1994; Sauer et al., 2004). In the process of the protein disaggregation it acts in the cooperation with the DnaK/Hsp70 systems (Glover & Lindquist, 1998; Zietkiewicz et al., 2006). Alone, the components of this bi-chaperone system exhibit no (ClpB) or weak (Hsp70) disaggregation activity. The mechanism of protein disaggregation by the ClpB/Hsp100 – Hsp70 bi-chaperone system requires the extraction of polypeptides from aggregates. The exposed unstructured regions of substrates are recognized usually by both, Hsp70 and Hsp100, chaperones. This reaction requires binding of ATP but not its hydrolysis. Next, the polypeptides are being unfolded by forcible translocation through the central channel of ClpB/Hsp100 by the aid of threading activity, common for other AAA+ proteins involved in proteolysis. The conserved aromatic residues located in the central pore of ClpB are crucial for the translocation of a substrate. The polypeptides are then released back into the cellular milieu in reaction requiring the hydrolysis of ATP. The released polypeptide may be subsequently refolded by the Hsp70 chaperone system or it refolds spontaneously (Lee et al., 2007; Lum et al., 2008). Protein Unfolding and Degradation Cell survival during various stress conditions depends on the efficient elimination of damaged or unfolded proteins. The correct balance between folding and degradation of misfolded proteins is ensured by protein quality control systems. They comprise both molecular chaperones, that refold proteins or rescue them from aggregates, and proteases, that degrade the irreversibly damaged proteins. The protein quality control system is also essential for maintaining the permissive cellular concentration of regulatory proteins, disassembly of protein complexes and turnover of properly folded proteins. In prokaryotic cells a number of specialized chaperone-assisted ATP-dependent proteases are used for turnover of misfolded proteins and the degradation of regulatory proteins (Zwickl et al., 2000). These include proteasome-like machines that consist of substrate specific component - an AAA+ protein and a proteolytic component.

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Proteolysis in the Bacterial Cytoplasm Proteolysis in the bacterial cytoplasm is mainly catalyzed by the ATP-dependent proteolytic machineries, termed the AAA+ proteases. The general purpose of their function is elimination of misfolded and damaged proteins from the cell. Their action represents a strategy to prevent accumulation of potentially toxic aggregates. Thus, the AAA+ proteases constitute a cellular protein quality control system, essential for environmental adaptation. On the other hand, the AAA+ proteases perform regulatory proteolysis as well. Degradation of regulatory proteins (e.g. transcription factors, signaling proteins) in their native state represents an intricate way to control key processes in cell cycle or pathogenesis (Schmidt et al., 2009).

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Structural Features of AAA+ Proteases In the bacterial cytoplasm the ATP-dependent proteolysis is performed by members of the caseinolytic AAA+ proteases: Clp, Lon and the membrane-bound FtsH protease. The AAA+ proteases constitute a family of several structurally related oligomeric complexes composed of two functional units with separate activities: a cylinder/barrel-like proteolytic core and hexameric chaperone rings which belong to the AAA+ superfamily of ATPases. The protease active sites are located in an inner proteolytic chamber of the proteolytic core and are sequestered from the external solution. The chaperone subunit is responsible for substrate recognition, unfolding and threading of the extended polypeptide chain to the proteolytic chamber. Substrate unfolding occurs by conformational changes of the chaperone and is driven by hydrolysis of ATP. Then, the substrates are actively translocated into the proteolytic chamber for degradation (Schmidt et al., 2009). In the Clp protein family two different proteolytic cores (ClpP and ClpY) and multiple chaperone rings (ClpA, ClpC, ClpE, ClpX, ClpY) are distinguished. This results in several possible Clp complexes. ClpP protease forms complexes with various chaperones: ClpA, ClpC, ClpE or ClpX. However, ClpY (HslU) interacts only with ClpQ (HslV) and forms ClpYQ (HslUV). In the Gram-negative bacterium E. coli only three Clp complexes have been found: ClpAP, ClpXP and ClpYQ (HslUV) (Figure 2a). The ClpCP and ClpEP complexes have been particularly characterized in the Gram-positive bacterium Bacillus subtilis (Kress et al., 2009). The ClpP protein is a conserved serine protease with the catalytic triad consisting of Ser111, His136 and Asp185. ClpP comprises two staged heptameric rings. Analysis of the crystal structure of the ClpP 14-mer carried out by Wang et al. (1997) suggests the mechanism of ClpP oligomerization in which the heptameric rings are formed first. Then they associate into a double-ring enclosing a roughly spherical hollow cavity of about 51Å in diameter with two axial pores of about 10Å. Inside the cavity 14 proteolytic sites are localized close to the equatorial plane of the ClpP barrel. In the ClpP monomer two distinct domains can be distinguished: a head domain, which comprizes the majority of the protein and forms the top of the tetradecamer, and a handle domain (consisting of β-strand and helix E), which forms

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(A) Figure 2. (Continued).

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(B) Figure 2. Structural features of AAA+ proteins. (a) Domain organization of Clp chaperones. The defined feature of AAA+ proteins is the presence of a basic core of 200-250 amino acids that comprise several conserved motifs including those necessary for binding and hydrolysis of ATP, the Walker A (A) and Walker B (B), respectively [Erzberger and Berger, 2006; Hanson and Whiteheart, 2005]. ClpA has two AAA domains (AAA1, AAA2) and belongs to class I proteins, whereas ClpX and ClpY/HslU possess one AAA domain, and are members of class II proteins. The nucleotide binding domains are highly conserved, whereas the N-terminal domains, present in ClpA and ClpX, have a low sequence homology. The single AAA domain of HslU shares homology to AAA2 domain of ClpA and is interrupted by an I-domain (I), proposed to bind SulA. (b) Domain organization of Lon and the membrane-bound FtsH protease. The AAA domain and the protease domain are fused together in a single polypeptide. Transmembrane domains (TM) of FtsH are located at the N-terminus. Lon is a serine protease and amino acid residues of catalytic dyad, serine and lysine, are indicated. FtsH is a metaloprotease and position of zinc-coordinating motif is marked [Zn(II)].

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Figure 3. Model of protein degradation by the Clp proteases. Clp chaperones form the homohexameric ring structures with a narrow central pore. The chaperone subunit associates with an oligomeric Clp protease subunit (a). The chaperone component recognizes, binds and unfolds a substrate using the energy of ATP hydrolysis (b, c). The unfolded polypeptide is actively translocated into the proteolytic component for degradation (d).

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the equatorial wall. The handle domain is also involved in the formation of the tetradecameric structure. The handle domains of one heptameric ring intercalate with the handle domains from the opposite heptameric ring (Wang et al., 1997). ClpP undergoes autocatalytic cleavage resulting in the release of a 14 amino acid propeptide. This reaction leads to activation of the protease ClpP (Maurizi et al., 1990). In the absence of the chaperone subunit ClpP displays limited peptidase activity and can perform only the hydrolysis of peptides of up to six amino acids (Gottesman et al., 1997). In E. coli ClpP is able to from complexes with ClpX or ClpA (Figure 2a). The association of the Clp protease with the chaperone subunit occurs only when the Clp ATPases are in their nucleotide-bound, hexameric forms. Although ClpA dimerizes in the absence of ATP, the formation of hexameric structure requires ATP. Binding of ATP induces the assembly of ClpA oligomer: starting from dimers via a tetrameric intermediate into the hexamer. The hexamer undergoes conformational changes to form active ATPase and is able to associate with the Clp protease (Kress et al., 2007). In contrast to ClpA, ClpX can from hexamer in the absence of the nucleotide. However, the assembly with the ClpP protease requires ATP. The presence of the nucleotide is also necessary for the interaction of ClpY chaperone with the protease ClpQ (Kress et al., 2009). General model of protein degradation by the Clp chaperone-protease system is presented in Figure 3. Unlike the Clp chaperone-proteases, where functional subunits are associated noncovalently, the Lon and FtsH proteases have the ATPase domains and proteolytic domains fused into the same polypeptide chain (Figure 2b). Data obtained by electron microscopy and crystallization of individual domains suggest that the E. coli Lon forms a functional homohexameric ring with a central cavity where proteolytic sites are isolated from the external solution. A general model of substrate degradation by Lon is consistent with the mechanism of substrate degradation by other AAA+ proteases. Each subunit of oligomeric Lon, mass of 87 kDa, possesses at least three domains (Figure 2b). The N-terminal domain exhibits the ability to bind proteins and is believed to be involved in substrate recognition and regulation of the protease activity. A central domain with a conserved ATP binding motif (GPPGVGKT) exhibits ATPase activity. The proteolytic domain, localized at the C-terminus, comprises amino acids residues of catalytic dyad: Ser679 and Lys722, sharing no homology to the classical catalytic triad of serine proteases (Rotanova et al., 2006). Lon and Clp chaperone-proteases are the typical cytoplasmic proteins, whereas FtsH is a membrane-associated protease (Narberhaus et al., 2009). The E. coli FtsH monomer, mass of 71 kDa, possesses transmembrane segments located at the N-terminal end, a central region with ATPase activity, comprising features specific for AAA+ proteins, and a metalloprotease domain containing the zinc-coordinating motifs (Figure 2b). FtsH forms a large membranespanning holoenzyme in complex with several prohibitin-like proteins, whose role is modulation of proteolytic activity. While Lon and Clp chaperone-proteases perform proteolysis of cytoplasmic proteins, FtsH protease degrades the misfolded or incorrectly inserted membrane proteins.

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Mechanisms of Substrate Recognition The general function of AAA+ proteases is the removal of misfolded or damaged proteins before they can form potentially deleterious aggregates. The cytosolic AAA+ proteases act synergistically. The E. coli cells lacking a single AAA+ protease are viable but exhibit several defects. For example, the lon- cells are sensitive to ultraviolet light as a result of stabilization of the SulA cell division regulator (Higashitani et al., 1997). Another defect is the overproduction of capsular polysaccharide caused by the stabilization of a positive regulator of capsule synthesis, RcsA (Torres-Cabassa & Gottesman, 1987). FtsH is the only essential protease in E. coli due to its critcal role in protein and membrane lipid homeostasis (Narberhaus et al., 2009). However, proteases ClpXP or Lon are essential for viability of E. coli cells at 420C when the level of the major E. coli chaperone DnaK is reduced (Tomoyasu et al., 2001). Moreover, the Lon protease is the most efficient protease in degrading misfolded proteins in the cells lacking DnaK. These findings represent an example of interplay between proteases and chaperones in protein quality control. It is still unclear how these enzymatic systems distinguish between unfolded proteins destined for refolding and damaged proteins destined for degradation and whether a particular protein should be directed for refolding or proteolysis. Mechanisms that allow the selective recognition of misfolded or damaged proteins by proteases prevent the accumulation of potentially toxic aggregates and ensure proper proteolysis but avoid the uncontrolled protein degradation. Changes of the environmental conditions, such as an increase in temperature over the physiological limit, can destabilize the structure of a protein. Proteins become partially or completely unfolded and the hydrophobic amino acid residues, buried inside proteins in their native conformation, become exposed. Identification of substrates is based on recognition of the long hydrophobic stretches of the polypeptide backbone, inaccessible in folded state. The recent studies by Gur and Sauer (2008) demonstrated that the Lon protease recognizes the hydrophobic peptide stretches enriched in the aromatic residues and the denatured polypeptides lacking such sequences are poor substrates. Among the AAA+ proteases Lon is responsible for approximately 50 % of the turnover of abnormal proteins in E. coli (Schrimer et al., 1996). Rosen et al. (2002) showed that E. coli cells lacking the Lon protease contain three times more aggregated proteins than the wild type cells. The tight control of proteolysis is also ensured due to the three-dimensional architecture of multimer of the AAA+ protease. The active sites of the proteolytic subunit are sequestered inside the barrel-like structure and can be reached only after passing through a narrow axial pore of chaperone subunit, the process that is forced by energy of ATP hydrolysis (Baker & Sauer, 2006). Several mechanisms of recognition of proteins destined for degradation have been identified. These mechanisms rely on the specific features of a target protein. The presence of the specific degradation signals, so called degrons, and their accessibility serve as an elegant mechanism to control stability of proteins under all conditions (Schmidt et al., 2009). A specific class of substrates comprises proteins with co-translationally attached SsrAtags to their C-terminal ends. The SsrA-tag consists of 11 amino acids encoded by a small rescue RNA molecule and becomes attached to a nascent polypeptide stalled on a ribosome. Such polypetides are often the products of the truncated mRNAs translation. As the result, the SsrA-tagged protein is degraded just after its release from the ribosome (Moore & Sauer, 2007). The SsrA-tagged proteins are generally degraded by ClpXP with the aid of SspB, also

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known as SprE or MviA. SspB is a small ClpX-specific adaptor protein which binds and delivers the SsrA-tagged proteins to the ClpX chaperone and enhances their degradation by ClpXP (Levchenko et al., 2000, 2003). SspB binds the SsrA-tagged protein at its N-terminus and docks to ClpX through a C-terminal binding motif. The recognition motifs of SspB and ClpX within the SsrA-tag are separated by a highly disordered stretch of approximately 40 amino acids which ensures an appropriate handover of the SsrA-tagged proteins to ClpX (Wah et al., 2003; Dougan et al., 2003). ClpA also possesses a structural motif for the interaction with the SsrA-tag. However, the chaperone ClpA is involved in a complex with ClpS, a specific adaptor protein of ClpA. This interaction represses the recognition of the SsrA-tagged substrates (Dougan et al., 2002). Under stress conditions, when SsrA-tagged proteins are abundant and ClpXP is overloaded, ClpAP is responsible for degradation of a significant amount of SsrA-proteins. Also, the SsrA-tagged proteins serve as substrates for ClpAP in the absence of ClpS (Maglica et al., 2008). Protease Lon and FtsH also degrade SsrA-taged proteins, however, compared to ClpXP and ClpAP, less efficiently (Choy et al., 2007; Lies & Maurizi, 2008; Herman et al., 2003). ClpAP in combination with ClpS is responsible for the degradation of substrates containing degradation signals at their N-terminal end (N-degron). The N-end rule states that the half-life of a protein depends on the N-terminal amino acid residue (Varshavsky et al., 1996; Mogk et al., 2007). In E. coli, several aromatic amino acids (Trp, Phe and Tyr) and Leu residues serve as the primary destabilizing residues. Proteins possessing one of these residues at their N-termini are recognized by ClpS, which directs them to degradation by the ClpAP complex. The N-terminal basic residues Arg and Lys serve as the secondary destabilizing residues. They are recognized by a leucinyl/phenylalanyl-tRNA-protein-transferase (L/F transferase) that catalyzes the attachment of the primary destabilizing residue, Leu or Phe, thus generating the N-end rule substrate with N-degron sequences FR, LR, FK or LK (Varshavsky et al., 1996; Mogk et al., 2007).

Proteolysis as a Control Mechanism under Stressful Conditions Proteolysis represents an important regulatory mechanism that allows a rapid adaptation of a cell to stressful conditions. It has been demonstrated that a sudden upshift of the growth temperature of the E. coli culture results in generation of denatured proteins. These proteins aggregate to form a distinct fraction, named the S fraction. In the wild type cells the S fraction exhibits a transient nature: the S fraction reaches its maximal size after 15 min at 45oC and disappears in 10 min following the transfer of the culture to 37oC (Kucharczyk et al., 1991). However, in the AAA+protease-deficient mutants a temporal or partial stabilization of the S fraction has been observed. In the cells lacking Lon or Clp proteases the S fractions were larger than in the corresponding wt cells. The disappearance of the S fractions from the protease defective strains was also retarded (Laskowska et al., 1996). Proteolysis plays also a key role in adjusting the level of several stress-related proteins. Under heat shock conditions expression of the heat shock genes is controlled by alternative factor sigma32 (RpoH) (Nonaka et al., 2006). The concentration of sigma32 is regulated by proteolysis that is performed by FtsH and to minor extent by other proteases (Blaszczak et al., 1999; Kanemori et al., 1999). At physiological conditions sigma32 is maintained at a low level

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of 50 molecules per cell (Strauss et al., 1987). The chaperone machineries DnaK/DnaJ and also GroEL/GroES participate in targeting of sigma32 for FtsH-dependent proteolysis. However, under stressful conditions the chaperones are engaged in binding of misfolded proteins; thus, sigma32 is able to bind to the promoters of the heat shock genes and initiates their transcription. Under heat shock conditions the concentration of sigma32 increases to 1000 molecules per cell (Strauss et al., 1987). The AAA+ proteases are engaged in a variety of regulatory functions which are shortly presented in Table 1. Table 1. Examples of miscellaneous functions of AAA+ proteases in E. coli AAA+ protease Clp chaperoneprotease

Cellular function Protein quality control (N-end rule, SsrAtagged proteins)

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SOS response (UmuD’, UvrA, RecN) Cell division (SulA)

Lon

Regulation of cell division (SulA) Regulation of capsule synthesis (RcsA) Turnover of anti-toxin proteins Acid resistance (GadE) SOS response (UmuD, UmuC, RecA, RuvB) Nutritional stress Oxidative stress response (SoxS)

FtsH

Protein quality control (SsrA-tagged proteins) Quality control of membrane proteins (SecY, YccA) Control of heat shock response (RpoH/sigma32) Regulation of superoxide stress response (SoxS) Regulation of the lysis/lysogeny decision of phage λ (λXis, λCII, λCIII) Biofilm formation

References Levchenko et al., 2000, 2003; Maglica et al., 2008 Pruteanu and Baker, 2009 Muffler et al., 1996; Pratt et al., 1996 Higashitani et al., 1997 Torres-Cabassa and Gottesman, 1987 Gerdes, 2000; Van Melderen et al., 2009 Pruteanu and Baker, 2009 Kuroda et al., 2001 Shah et al., 2006 Narberhaus et al., 2009

Role of Molecular Chaperones and Proteases in the Cytoplasmic Inclusion Bodies Processing E. coli continues to be the most commonly used organism for production of recombinant proteins that do not require posttranslational processing. Numerous genes encoding prokaryotic, viral, or eukaryotic proteins have been cloned and expressed in bacterial cells.

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The expression of the foreign genes in E. coli achieves frequently levels of 10-15% of the total cell proteins. Such high-level production may lead to protein aggregation and formation of intracellular electron-dense insoluble deposits, called inclusion bodies (IBs) (Schlieker et al., 2002; Ventura & Villaverde, 2006). Inclusion bodies are homogeneous in composition. About 90% of total IBs proteins is the recombinant protein showing various conformations. There is an evidence that the proteins trapped in IBs can be partially structured and the biological activity of enzyme-based IBs can even be detected (Carrio& Villavede, 2002; Kuczynska-Wisnik et al., 2004). IBs are mainly formed from the folding intermediates which expose hydrophobic patches. These hydrophobic surfaces are not sufficiently protected against nonspecific interactions by the housekeeping molecular chaperones whose limited amounts are not sufficient to assist folding of the overproduced proteins (Mogk et al., 2002). Although IBs mainly consist of recombinant proteins, they contain also membrane proteins, phospholipids, nucleic acids and folding assistant proteins such as small heat shock proteins IbpA and IbpB, and main chaperones DnaK and GroEL (Allen et al., 1992; Carrio & Villaverde, 2002; Carrio & Villaverde, 2005). Deposition of proteins in the form of inclusion bodies was shown to be a reversible process (Carrio & Villaverde, 2002; Carrio & Villaverde, 2005; Schlieker et al., 2002; Ventura & Villaverde, 2006). In vivo, IBs can be disintegrated in few hours after protein synthesis arrest (Carrio & Villaverde, 2001). It was proposed that IBs are reservoirs of protease-resistant protein, available for folding or proteolysis under the favorable conditions. In many cases the formation of IBs is beneficial for protein purification. First, the inclusion bodies consist mainly of the protein of interest and are easily isolated by centrifugation. Second, the proteins that are easily degraded by proteases or are toxic to the host cells can be obtained in large quantities only in the form inclusion bodies. However, to recover a native protein from IBs, the in vitro refolding procedure should be applied and renaturation yields are often low (Baneyx, 1999; Ventura & Villaverde, 2006). Several methods have been suggested to improve solubilization and refolding of the overproduced proteins or to decrease the aggregation level in the host cell. One of these methods is optimalization of the folding process of an overproduced protein in vivo by the co-expression of molecular chaperones. It was shown that co-production of the molecular chaperone systems (IbpA, IbpB, ClpB, DnaK and GroELsystems) with the client protein can reduce deposition of the recombinant protein into inclusion bodies and improve the de novo folding of recombinant protein (Thomas and Baneyx, 1997; Carrio & Villaverde, 2001; de Marco et al., 2007). However, the effect of the chaperones co-expression on recombinant protein solubility depends on the nature of the overproduced protein and in some cases chaperones may even negatively affect the solubility and stability of a product (Lee & Olins, 1992). Vera et al. (2005) showed that Lon and ClpP proteases participate in the physiological disintegration of cytoplasmic bacterial inclusion bodies and in the absence of these proteases the removal of an aggregation prone β-galactosidase fusion protein used as a model was reduced up to 40%. While protease Lon participates in the disintegration process to a minor extent, the Clp protease appears as a main processor of aggregation-prone proteins. Clp degrades polypeptides physiologically released or releasable from IBs (Vera et al., 2005). Thus the recovery of recombinant proteins from IBs could be improved in strains deficient in the major cytoplasmic proteases. However, recent studies by Jurgen et al (2010) showed that the major ATP-dependent cytoplasmic proteases Clp and Lon, and also FtsH are not involved

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in quality control of recombinant proteins sequestered in form of IBs. They found that the αglucosidase of Saccharomyces cerevisiae which accumulates during overexpression in E. coli excusively in form of IBs is partially fragmented. However, none of these major cytoplasmic proteases are responsible for fragmentation, which occurs before aggregation.

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The Extracytoplasmic Protein Quality Control Systems The cell envelope of Gram negative bacteria surrounds the cytoplasm and consists of the inner (cytoplasmic) membrane, the periplasm with the murein sacculus, and the outer membrane (OM). It is the most exposed to the environment part of a cell, therefore it acts as the first barrier against the impact of the external environment. All the same, the periplasmic space is particularly prone to damage by the extracellular stress factors due to a low selectivity of the porous outer membrane (Oliver, 1996). For these reasons the extracytoplasmic protein quality control is a very important process that provides cellular homeostasis and survival under stressful conditions. The periplasm is a compartment that differs significantly from the cytoplasm. There is a lack of ATP, a reduced protein mobility and a more oxidizing environment compared to the cytoplasm. Accordingly, a separate protein quality control system has evolved in the periplasm. The polypeptides newly translocated to the periplasm are subject to folding and posttranslational modifications, and eventually formation of proper quaternary structures (Figure 4). Following the cleavage of the signal peptide, the polypeptides become substrates for the folding factors. This group comprises: (1) disulfide oxidoreductases, responsible for formation of proper disulfide bonds (Dsb proteins); (2) peptidyl-prolyl isomerases (PPI-ases), that catalyze the cis-trans isomerisation of the peptide bonds preceding proline residues (SurA, PpiA, PpiD, FkpA); (3) chaperones, that stabilize nonnative conformations of target proteins, prevent their aggregation and facilitate their folding (Skp, acid chaperones, PapD superfamily). Under physiological conditions a concerted action of these proteins leads to formation of correct, functional structures of extracytoplasmic proteins. However, under certain stressful conditions (e.g. heat shock, changes in osmolarity, redox stress, overproduction of recombinant proteins) the efficiency of this system may be not sufficient and misfolded proteins may accumulate. The presence of misfolded polypeptides in the cellular envelope becomes a signal to induce at least two different extracytoplasmic stress response systems: Cpx and sigma E (σE). Activation of these systems leads to an increased synthesis of periplasmic stress proteins, including chaperones and proteases (for review see Duguay & Silhavy, 2004; Miot & Betton, 2004; Dorel et al., 2006). Of these, the chaperone Skp, PPI-ase FkpA, and the protease HtrA (DegP) are particularly involved in the suppression of extracytoplasmic protein aggregation. Additionally SurA, a PPI-ase which is not a member of the σE or Cpx regulons , helps to maintain the periplasmic proteins in a soluble state. Under acid stress conditions, the chaperones HdeA and HdeB are responsible for protection of denatured proteins from aggregation and their subsequent refolding.

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M Mogensen & Otzzen, 2005; Duguuay & Silhavy, 2004; Bos & Tomassen, T 2004.

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Fiigure 4. Extracyttoplasmic protein quality control. Thhe precursors off extracytoplsmicc proteins are traanslocated across the inner mem mbrane (IM) throuugh a deedicated protein complex (“transslocon”). In the periplasmic p spacce they become substrates s for protein folding faactors that preven nt aggregation annd nonspecific interactions of thhe newly transloocated polypeptiddes (proteins accting as chaperon nes, e.g. Skp, SuurA), introduce proper p disulfide bonds b (Dsb prottein family), perfform ciis/trans isomerizzation of peptide bonds (PpiA). The T precursors of o outer membranne proteins (OM MPs) on their w to the outer membrane way m (OM)) are protected byy chaperones and kept in the insertion-competennt coonformation, beffore they becomee inserted into thhe phospholipid bilyer with the aid a of a speciallyy dedicated chhaperone compleex Omp85. The polypeptides p whhich do not fold properly p or lose their native connformation beecome substratess for proteases.

S Skp i many Gram m-negative Skp is thee best charactterized periplaasmic chaperoone, present in baacteria. Skp iss not an essenntial protein; however h E. cooli skp mutantts are characteerized by a reeduced level of o properly foolded outer meembrane proteeins (OMPs) (Chen ( & Hennnig, 1996). B Bacteria additio onally depriveed of htrA genne, coding forr periplasmic protease, p are viable v only att temperaturess below 37 oC and accumulaate nonnative OMPs O in the periplasm p (Schhafer et al., 19999). Skp is under u the contrrol of both thee σE and Cpx stress responsse system (Darrtigalongue ett al., 2001). Outer O membraane proteins, like l OmpA, OmpF, O PhoE and a LamB, arre the main suubstrates for Skp. S Apart froom the OMPss, also solublee periplasmic proteins weree shown to biind to Skp. These T include natural periplasmic proteinns Mal E andd OppA (Jarchhow et al.,

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2008), but also lysozyme or antibodies fragments (Bothman & Pluckthunn,1998, Walton & Sousa, 2004) . Mature Skp from E. coli is a strongly basic, 15.7 kDa protein. According to the crystal structure Skp is a homotrimer and shows a “jellyfish-like” architecture, whose “tentacle-like” α-helical protrusions form a cavity-like structure to bind substrate proteins (Walton & Sousa, 2004). The Skp molecule possesses asymmetric charge distribution with the negatively charged core domain and positively charged outer parts at the end of the tentacles. The polar nature of Skp helps the molecule to adapt the right orientation and directs it to the negatively charged outer membrane surface. This feature of Skp facilitates delivery of OMPs to their final destination. The inner cavity contains hydrophobic stretches and it is likely that hydrophobic residues of the transmembrane domains of the OMPs are bound at these locations. Transported OMP molecules contain also exposed negative charges; thus electrostatic interactions can play important role in stabilization Skp-OMP complexes (Qu et al., 2007). Interactions with Skp protect OMPs from aggregation during their passage to outer membrane. The in vitro experiments demonstrated that Skp prevents the aggregation of OmpA by forming a stable complex, which can be isolated by size exclusion chromatography. During the passage across the periplasm the unfolded hydrophobic β-barrel transmembrane domain of OmpA is protected within the inner cavity of Skp, whereas the periplasmic OmpA domain protrudes outside the Skp chaperone and folds into its native conformation (Walton et al., 2009).

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SurA Many Gram negative bacteria have a second periplasmic chaperone, Sur A, which is involved in delivery of OMPs to outer membrane (Duguay & Silhavy, 2004). E.coli cells deprived of surA or skp genes are viable, but a double deletion produces a synthetic lethal phenotype (Rizzitello et al., 2001). This observation suggests that these chaperones have overlapping functions in E. coli. Similarly to the skp mutants, the E. coli cells lacking SurA show reduced levels of correctly folded outer membrane proteins (LamB, OmpA, OmpC, OmpF) (Duguay & Silhavy, 2004; Lazar & Kolter, 1996). SurA belongs to the parvulin class of PPI-ases, and exhibits both chaperone and PPI-ase activity, localized in separated domains of SurA. However, the PPI-ase activity is rather weak and the E.coli mutants expressing SurA lacking PPI-ase activity show no defects in OMPs’ biogenesis (Behrens et al., 2001). Contrary to Skp, SurA is rather not involved in folding of soluble periplasmic proteins (Duguay & Silhavy, 2004). Studies with the peptide libraries indicated that SurA binds preferentially to the peptides containing the amino acid sequence aromatic-random-aromatic (Ar-X-Ar) and such motifs are found frequently in OMPs (Bitto & McKay, 2003, Hennecke et al., 2005, Xu et al., 2007). For example, OmpG contains 13 Ar-X-Ar motifs and OmpF – 7 motifs (Bitto & McKay, 2004). However, whereas SurA binding to small peptides has been demonstrated, the mechanism of protecting β-barrels from aggregation remains unclear.

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FkpA It is another periplasmic protein exhibiting dual activity: that of PPI-ase and chaperone (Ramm & Pluckthun, 2000, Arie et al., 2001) and its expression is activated via the σE pathway (Missiakas et al. 1996). However, contrary to SurA, it seems to be specific for soluble proteins (Bothmann & Pluckthun, 2000) and most probably plays important role at early stages of periplasmic proteins folding (Ramm & Pluckthun, 2000, Ramm & Pluckthun, 2001). FkpA functions as a homodimer. The shape of its molecule resembles the letter “V”, where the N-terminal chaperone domains form the bottom and the C-terminal PPI-ase domains are stretching outside. A space between the subunits is most probably responsible for binding of substrates (Saul et al., 2004). Interactions of FkpA and substrates are transient, since no stable FkpA-substrate complexes were isolated (Ramm & Pluckthun, 2000). Nevertheless, FkpA efficiently prevents aggregation of overexpressed mutated proteins or recombinant proteins exported to the periplasm (Ramm & Pluckthun, 2000, Arie et al., 2001).

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Acid-Stress Chaperones HdeA and HdeB Enteropathogenic bacteria, when travelling through the gastrointestinal tract, encounter the acidic environment of mammalian stomach (pH 1-3). Due to the permeability of outer membrane to molecules smaller than 600 Da (Koebnik et al., 2000), the periplasm is probably more vulnerable to acid stress than the cytoplasm. Recently, two periplasmic chaperones, HdeA and HdeB, which support the acid resistance in E. coli and Shigella flexneri, have been discovered (Gajiwala & Burley, 2000; Waterman & Small, 1996). Both proteins are needed to protect bacterial cells at low pH as the hdeA or hdeB bacteria are significantly less viable at pH values below 3. HdeA and HdeB have the ability to bind and protect from aggregation the acid denatured proteins, however they display different pH specificities. At pH 2 HdeA is mainly responsible for keeping the periplasmic proteins in the soluble form; HdeB at these conditions is much less efficient. At pH 3 HdeB is a better suppressor of aggregation that HdeA; however, certain substrates require a concerted action of both proteins for a maximal protection (Kern et al., 2007). The Hde proteins represent a very interesting example of the ATP-independent chaperones. To fulfill their protective roles they appear to utilize the natural host physiology: the external pH changes enable activation, inactivation and substrate-protein refolding. Under conditions physiological for bacteria HdeA and HdeB do not exhibit chaperone activities. Their molecules form homodimers and have an ordered structure with low surface hydrophobicity. When the enterobacteria reach acidic stomach, the structures of Hde proteins become disordered, dimers dissociate and the hydrophobic regions become exposed. In this state the acid chaperones show the highest affinity towards their substrates, bind the aciddenatured proteins and prevent their aggregation (Hong et al., 2005, Kern et al., 2007). The hydrophobic residues of HdeA involved in the substrate binding are located at the dimer interface, therefore monomerization seems to be a crucial step of HdeA activation. Owing to the partially unfolded state, HdeA has a highly flexible architecture and this feature seems to contribute to HdeA’s ability to adaptively bind to a wide range of different substrate proteins (Tapley et al., 2009). In the small intestine the pH is typically around 7; therefore HdeA and HdeB return to their ordered inactive forms and release their substrates which subsequently

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may be refolded or eventually degraded by the proteases that function at the physiological pH. It is proposed that the release of substrates occurs slowly and this feature may provide a convenient way to keep the concentration of unfolded polypeptides low. Because aggregation is strongly dependent on the concentration of aggregation-sensitive folding intermediates, the process that decreases concentration of such intermediates should suppress formation of aggregates and thereby promote refolding. It was demonstrated that HdeA can independently facilitate the refolding of acid-denatured alkaline phosphatase and other substrates to their catalytically active state in an in vitro assay (Tapley et al., 2010). That HdeA does not require additional chaperones in vitro, does not exclude the possibility that other periplasmic chaperones may assist HdeA in vivo. HdeA and HdeB can also form mixed aggregates with proteins that failed to be solubilized at the acidic pH and allow their solubilization at neutral pH. The aggregates formed in the presence of Hde are characterized by lower molecular weight and lower surface hydrophobicity as compared to the aggregates lacking the Hde chaperones. Moreover, during the recovery of acid-stressed bacterial cells at neutral pH, HdeA and HdeB assist in the solubilization and renaturation of the aggregated proteins. These include maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ (Malki et al., 2008).

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Periplasmic Proteases HtrA HtrA is a heat shock protein whose presence is indispensable for cell survival under certain stress conditions. It is required when the cells are exposed to temperatures higher than 42oC (Lipinska et al., 1989), in the presence of oxidative agents affecting cellular envelope (ex. ferrous ions) (Skorko-Glonek et al., 1999) or when the misfolded OMPs are accumulated within the periplasm (Misra et al., 2000).The htrA gene’s expression is strongly stimulated under stress conditions affecting cellular envelope and is controlled by two overlapping, but distinct pathways: σ24 and Cpx (Clausen et al., 2002). HtrA was shown to combine two activities: proteolytic and chaperone, which are switched in the temperature-dependent manner (Spiess et al., 1999). For these reasons HtrA seems to be a key player in the extracytoplasmic protein quality control system (Duguay & Silhavy, 2004).

Characterization of the Proteolytic Activity of HtrA HtrA is an endopeptidase that that does not require the presence of ATP and shows a pH tolerance over a broad range (Lipinska et al., 1990). The proteolytic activity shows a strong temperature dependence. In the in vitro assay using β-casein as a substrate no cleavage is observed below 22oC. At temperatures above 30oC, the proteolytic activity rapidly increases in a nonlinear fashion to reach the maximum approximately at 50oC (Spiess et al., 1999; Skorko-Glonek et al., 1995). The strong stimulation of activity at elevated temperatures is typical for heat shock proteins and enables them to cope with the stress effects. The shift of temperature to 42oC (and above) leads to protein misfolding. It is believed that HtrA is a primary protease responsible for the removal of the abnormal or denatured proteins within the cellular envelope. It was shown that the lack of functional htrA gene

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results in the increased accumulation of protein aggregates upon heat shock conditions. Furthermore, when the cells return to the physiological temperature, the removal of these aggregates is delayed in time compared to the wild type strain (Laskowska et al., 1996). The known polypeptide substrates comprise: hybrid proteins (Strauch & Beckwith, 1988), recombinant proteins (Baneyx & Georgiou, 1991), misfolded periplasmic proteins such as maltose binding protein (MBP) (Betton et al, 1998), PhoA (Sone et al, 1997) and MalS (Spiess et al., 1999), misfolded subunits of outer membrane complexes (OmpF, OmpC or major pilin subunit PapA) (Misra et al., 2000, CastilloKeller & Misra, 2003; Jones et al., 2002). In vitro it degrades unstructured proteins such as casein (Lipinska et al., 1990), heat denatured proteins such as citrate synthase and malate dehydrogenase (Kim et al., 1999), reduced polypeptides such as lysozyme, α-lactalbumin or insulin (Skorko-Glonek et al., 2003, Kim et al., 1999). All these substrates share a common feature: they are at least partially unfolded. HtrA in general does not degrade stably folded native proteins like MalS (Spiess et al., 1999), BSA, ovalbumin, globin or insulin (Lipinska et al., 1990). The reason for this selectivity is the cleavage-site preference. HtrA preferably hydrolyzes the peptide bonds which follow hydrophobic residues: Val or Ile (Kolmar et al., 1996). Such residues are usually buried within the hydrophobic core of the protein and thus are inaccessible in the native state.

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Characterization of the Chaperone Activity of HtrA Aside from its proteolytic activity HtrA was shown to exhibit an additional, chaperone activity (Spiess et al., 1999). Specifically, HtrA was found to be involved in the MalS αamylase folding. The chaperone-like activity of HtrA was also reported in relation to the mutated outer membrane proteins OmpF and OmpC (Misra et al., 2000, Castillokeller et al., 2003). The mutated Omp proteins were defective in the proper assembly and their overexpression in the htrA background was lethal for the cells. The mutant Omp-mediated lethality in the htrA background was reversed by overproduction of the mutant HtrAS210A, which is proteolytically inactive but retains its chaperone activity. However, the proper assembly of the mutant Omps in the outer membrane did not occur, indicating that HtrAS210A did not reverse the lethal effect of the mutated Omps by correcting their assembly but rather by capturing them and in this way removing them from the assembly pathway. The importance of the chaperone activity of HtrA was confirmed by the in vivo experiments showing the synthetic phenotypes of null mutations in htrA and surA (Rizzitello et al., 2001). The htrA surA combination was bactericidal and caused lethality of cells at temperatures above 23oC. Expression of the mutated htrAS210A gene from a plasmid or chromosome allowed growth at temperatures up to 37oC; hence, the synthetic phenotype of the double mutant strain could not be attributed to the loss of the HtrA protease activity. In the recent work (Skorko-Glonek et al., 2007) it was demonstrated that HtrA protein may act as a chaperone not only at low temperatures but under the conditions of heat shock as well. The high levels of proteolytically inactive HtrA S210A were able to rescue the temperature-sensitive phenotype of the htrA mutants. In the htrA strain the lack of viability upon heat shock was accompanied by the high content of aggregated proteins, whereas the overproduction of HtrAS210A in this strain resulted in the decrease of the amount of large aggregates and improved viability at elevated temperatures. Hence, the tuning of the level of

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protein aggregation may be an important role for HtrA at stress conditions. This prediction was further supported by the analysis of growth pattern and susceptibility to accumulate protein aggregates by the double mutant strain B178htrAdsbA subjected to heat shock. DsbA protein is the major periplasmic oxidase responsible for introducing disulfide bonds to the extracytoplasmic proteins (Nakamoto & Bardwell, 2004). Proteins lacking their disulfide bonds are less stable and often fail to achieve proper structure; thus, are susceptible to aggregation. Therefore, although the single mutant dsbA strain did not show any growth defects at elevated temperatures, in the double mutant dsbAhtrA the effects of heat shock, especially the content of aggregated proteins, were much more pronounced. In this case the overproduction of HtrAS210A was not sufficient to provide viability of the bacterial cells. Although in the presence of HtrAS210A the amount of large aggregates in the strain dsbAhtrA was significantly diminished, the remaining aggregation events must have caused effects toxic for the cells (Skorko-Glonek et al., 2007). To perform such a protective function in the cell HtrA must be capable of very efficient binding of the damaged proteins. Indeed, the results of the in vitro experiments using chemically denatured lysozyme proved that HtrA was able to bind the unfolded polypeptides and prevent the formation of large aggregates over a wide range of temperatures (30 - 45oC). HtrA seems to act by restricting the size of aggregates to smaller particles and keeping them in the soluble form (Skorko-Glonek et al., 2007). The chaperone activity is independent from the proteolytic activity. The HtrA S210A mutant lacking the proteolytic activity retains its chaperone activity (Spiess et al., 1999). Moreover, the recent work of Jomaa et al (2007) implies that HtrA recognizes substrate molecules targeted for degradation or refolding in a different manner. In particular, for the proteolytic activity the presence of PDZ1 domain is required whereas for chaperone activity the PDZ domains are dispensable and the mechanism of binding the substrates suitable for refolding is not known (Iwanczyk et al., 2007).

Other Extracytoplasmic Proteases The remaining periplasmic and membrane proteases are less well characterized and their physiological roles are not fully understood. Nevertheless, the proteases Tsp (Prc) and Ptr are potentially engaged in the extracytoplasmic protein quality control, as the proteolysis of recombinant proteins was shown to be lowered in E. coli strains devoid of these proteases (Meerman and Georgiou, 1994). Tsp (tail specific protease) is an endoprotease selective for the proteins that expose nonpolar C-terminal sequences, especially: A (or L)-A (or Y)-A (Keiler & Sauer, 1996). This kind of sequences are normally not present in proteins; however, they can be added to polypeptides as the result of the SsrA-tagging system. Ptr (Pi, protease III) is a metaloprotease initially shown to degrade polypeptides such as insulin, glucagon or β-galactisidase fragments. However, it can also recognize large proteins of abnormal structure. For example, it was demonstrated that PtrA degrades a fusion protein, protein A-β-lactamase, in an in vivo assay (Keiler & Sauer, 2004). Whether the proteases Tsp and Ptr can contribute to the suppression of protein aggregation in periplasm is up to date not known.

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Expression of Recombinant Proteins in the Periplasm – Troubleshooting The periplasm is a compartment alternative to the cytoplasm to accumulate overproduced recombinant proteins. It holds a significant biotechnological importance in the production of secreted proteins that require post-translational modifications, such as signal sequence processing, formation of proper disulfide bonds, covalent heme linkage, proteolytical processing and prolyl cis-trans isomerization (Stampolidis et al., 2009). Moreover, the periplasm consists of approximately only 5% of the total cellular protein. This feature may facilitate the purification of the proteins exported to the periplasm, as the target protein can be easily isolated from the majority of the cytoplasmic proteins (Oliver, 1996, Narayanan et al., 2008). However, in the periplasm, like in the cytoplasm, overproduction of proteins may lead to their aggregation and formation of inclusion bodies (Arie et al. 2006). To overcome this problem several approaches are undertaken. One of the promising ones is a simultaneous expression of the periplasmic folding factors, especially Skp and FkpA. It has been reported that co-expression of Skp with a protein of interest yields a more soluble and active form of the protein in E. coli. The most important examples comprise antibody fragments (Mavrangelos et al., 2001) and mammalian transcriptional factors Sox2 and Nanog (Ha 2009). The two latter proteins were shown to be stabilized by tight interaction with Skp. Sox2 and Nanog were subsequently purified as stable complexes with Skp, which retained their ability to bind their cognate DNA sequences (Ha et al., 2009). FkpA was shown to stabilize a number of intrinsically unstable proteins, which are prone to misfolding and aggregation. For example elevated level of FkpA allows expression of soluble single chain T cell receptors (scTCR). The final protein yields were shown to be comparable to the previously obtained periplasmic expression systems, but they were achieved without fusions or amino acid modification of the TCR moiety (Gunnarsen et al., 2010). Many eukaryotic proteins contain disulfide bonds which often are indispensable for stabilization of native conformation. In such case a co-expression of oxido-reductases of Dsb family (e.g. DsbA or DsbC) may be helpful (Sorensen & Mortensen, 2005).

Conclusion The bacterial protein quality control systems are the primary line of defense against consequences of stressful conditions. Due to their concerted action they prevent accumulation of damaged and misfolded proteins. This goal is achieved by holding and refolding of proteins with non-native structure (chaperones) or degradation of irreversibly damaged proteins (proteases). These features of elements of protein quality control systems make them valuable tools for efficient expression of heterologous proteins in bacterial systems. However, in spite of many years of extensive research, several questions remain open. First, the exact mechanisms of action of quality systems are not yet completely understood. Second, how a decision “to degrade or to refold” – is made? Another interesting question is

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how the periplasmic chaperones perform protein folding in the absence of ATP and what is their alternative energy source. Further research should bring answers to these questions.

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In: Protein Aggregation Editor: Douglas A. Stein, pp. 79-109

ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 3

Native Functions of Amyloid Reeba S. Jacob, A. Anoop, Pradeep K. Singh and Samir K. Maji* Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400 076, India

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Abstract Amyloids are highly ordered protein/peptide aggregates with cross-β-sheet rich structure. Amyloids are originally associated with many neurodegenerative diseases including Alzheimer's, Parkinson's and Type II diabetes. The natively structured or unstructured proteins adopt partially folded conformation and subsequently selfassociates through nucleation dependent polymerization to form amyloid fibrils. These fibrils are very stable, resistant to proteases and to harsh environmental conditions. Recently, several studies have indicated that amyloid fibrils are also abundant in living organisms from prokaryotes to eukaryotes, where amyloids are evolved to perform native functions of the host. Such amyloids are termed as ‘functional amyloids’. Curli in E. coli and Het-s in Podospora anserina are well known examples of functional amyloids in bacteria and fungi respectively. Yeast prions do not cause cell death rather help the host to survive in certain environmental conditions. In mammals, Pmel17 forms amyloid inside the melanosome, where it is involved in skin pigmentation. Moreover, recent studies have suggested that peptide/protein hormones in pituitary secretory granules are stored in amyloid-like aggregates. In this chapter, we summarize the recent discoveries of functional amyloids, where amyloid fibrils are evolved for an organism's survival rather than creating only diseases.

Introduction Amyloids are protein aggregates historically associated with many diseases. More than 20 human diseases including Alzheimer's, Parkinson's, prions and Type II diabetes are *

Correspondence should be addressed to Samir K. Maji, [email protected]

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associated with protein aggregation and amyloid fibril formation [1]. Amyloid fibrils are composed of cross-β-sheet rich structure, where the individual strands of each β-sheet run perpendicular to the fibril axis (4.7 Å spacing) whereas the β-sheets (~10 Å spacing) themselves are parallel to the fibril axis [2] (Figure 1). Due to cross-β-sheet arrangement, amyloids can bind to β-sheet structure specific dyes such as Thioflavin T (ThT) [3] and Congo red (CR) [4]. It also provides birefringence after binding to CR and visualized under cross-polarized light. Generally, amyloids are of 2-10 nm in diameter and several micrometers long and composed of 2-4 protofilaments, in which individual protofilaments are further associated by lateral fashion or twisted-helical fashion [5,6] (as shown in Fig 1 and 2). Both natively structured and natively unstructured proteins/peptides can form amyloid fibrils through a partially folded intermediate [7] and subsequently formation of the small oligomers and protofibrils (Fig 1). Amyloid formation most often occurs via a nucleation dependent polymerization reaction [8], where preformed amyloids (‘seed’) can recruit the soluble protein counter part and make them amyloid and continue to grow (Figure 2). Recently, it has been suggested that proteins that are not apparently associated with amyloid diseases can also form amyloid-like fibrils in vitro [9,10]. The fibril formations by non-disease associated proteins lead to the belief that the formation of amyloid fibrils is a generic property of the polypeptide chain [11]. Moreover, the ability of amyloid formation by a large group of proteins with diverse amino acid sequence has further strengthened the above hypothesis that (m)any protein(s) can form amyloid under appropriate conditions. The amyloids are noncrystalline and insoluble in water, precluding their structure determination by conventional Xray crystallography or solution-state nuclear magnetic resonance (NMR). However, recent advancements in solid-state NMR and synchrotron X-ray crystallography has made it possible to elucidate the structure of amyloids from various proteins and peptides. These studies have further supported the cross-β-structure in amyloid fibrils [2]. These studies have also led to a unified structural scheme of steric zipper of all diseases and functional amyloids [12-15]. Furthermore, in line with recent suggestions that amyloid fibrils are less toxic compared to its precursor soluble aggregates [16,17] and the discovery of several functional amyloids [18-20] (Table 1) in nature suggest that engineered amyloid by manipulating peptide self-assembly could be useful for designing scaffold for bioactive materials and tissue engineering [21-24].

1. Functional Amyloids Although amyloids are known to be associated with many diseases, several examples exist where it has been shown that amyloid formation has evolved in nature to perform proper function of host organism (Table 1) [1,18,25]. Bacteria such as Escherichia coli and Salmonella spp. produce proteinaceous, extracellular, amyloid fibrils called curli that are involved in surface and cell-to-cell contacts that promote community behavior and host colonization [26]. The eggshell protein (chorion) of silkworm forms amyloids that have been reported to protect the oocyte and the developing embryo from a wide range of environmental hazards [27]. Yeast prions and Het-s proteins are well-studied examples of functional amyloids in fungi. The yeast prions such as Sup35 and Ure2p, the amyloid form of these proteins are important for the survival of host, rather than causing the cell death [28,29]. The HET-s protein of filamentous fungus Podospora anserina forms infectious amyloid that

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trrigger a complex programm med cell deathh phenomenonn (heterokaryoon incompatibbility) [30]. Jeeff Kelly and his group disccovered the fiirst mammaliaan functional amyloid, wheere amyloid fibrils of Pmell17 inside thee melanosomee appear to prrotect melanocytes from haarm during m melanin syntheesis [31]. Reecently, it haas been sugggested that piituitary horm mones form am myloid-like sttructure, whicch is importannt for hormonne storage inn secretory granules and reelease [32]. In nterestingly, protein p aggreegates within the inclusionn bodies also appear to coontain amyloid-like structurre. Thus amylloid aggregation in inclusioon body formaation might bee a mode of detoxificationn mechanism during d heterologous proteinn expression in bacteria [333]. In the ligh ht of functionnal amyloid, designing d amyyloid-based funnctional materrial will be prromising for applications a annd advanced reesearch in nanno-biotechnoloogy [23].

Fiigure 1. Schemattic representation of possible follding pathway(s)) of an endogenoously synthesizeed protein. In adddition of protein n folding to nativve structure, prootein can aggregaate into amorphoous state (devoidd of peersistent structurre) or into small oligomers. Thesse small soluble oligomers (IV) can c transform innto higher orrder oligomers of o "pore" (V) andd/or worm-like "protofibrils" (VII) and ultimatelyy to β-sheet rich amyloid fibbrils. The unfold ded protein (I), right r after the synnthesis could forrm partially foldded intermediatess (II and III). A of these differrent conformatioonal state and theeir conversions are All a tightly regulaated in the biologgical system byy the help of pro otein homeostaticc machinery suchh as molecular chaperones, c proteosomal degradaation and quuality control pro ocesses. Amyloiid fibrils have chharacteristic physical properties: It shows fibrillaar m morphology under EM (D), can bind to Congo redd and gives bireffringence under cross-polarized light (A), Xraay powder diffracction (C) of the amyloid fibrils generally g producce two reflectionns at ∼4.7 Å (merridional, chharacteristic of distance d betweenn two β-strand) and a ∼10.2 Å (equuatorial, distancee between two β-sheets) that is characteristic off cross-β-sheet structure s (B).

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Fiigure 2. Protein aggregation a intoo disease and funnctional amyloidd. (A) Amyloid formation f by nuccleation deependent polymeerization mechannism showing innitial lag phase and a then exponenntial growth and stationary phhase. Most of thee functional and disease amyloidd formation folloow this mechanism. Preformed amyloid a fibbrils can acts as a seed to accelerate the kinetics of fibril formatiion by reducing the lag time (Unnseeded and seeeded polymerizations are repressented in solid annd dotted lines, respectively). r (B B) Morphology of o amyloid from α-synuclein (associated withh Parkinson's dissease), Aβ42 (asssociated with Alzheimer's diseaase), hCRF ociated with horrmone storage) showing 2-3 filam ments are associated to form higgher order annd hGRF40 (asso am myloid fibrils. Sccale bars are 5000 nm.

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2 Bacte 2. erial Am myloid val of bacteriaa depends on their t interactioon with the environment thhat is often The surviv mediated by the presencee of cell-suurface organeelle enrichedd of protein polymers m [220,26,34,35]. These T proteinn based polym mers are impliccated for varioous functions of bacteria suuch as attach hment to thee surfaces, locomotion l a and host-pathhogen interacttions. The exxtracellular en nvironment might m be very harsh due to high salt concentrations, non-optimal n pH H, temperaturre and presennce of chemicals, where poolymeric proteein fibrils hellp bacterial suurvival. The extracellular e p protein based fibrils formattion should thherefore be sppontaneous annd evolutionaary optimized.. The electronn microscopy (EM) and attomic force microscopic m sttudies have reeported the am myloid naturee of these fibbrils [20,26]. One O of the well-studied w fuunctional amy yloid is curli fibrils of the gram-negativve bacteria is E. coli [26,366]. Several otther enteric bacteria b such as Salmonellaa spp. have also a been show wn to producce fimbriae knnown as tafi, which have amyloid-like structure. Thhe other familly of bacteriaal amyloids knnown as Chap plins was founnd in the gram m-positive filam mentous Strepptomyces coeliicolor [20]. C Chaplin amylo oids are involvved in produuction of aeriaal hyphae andd dispersal off spores in filamentous baacteria. Severral other funcctional amylooids in bacteria including pili from M Mycobacterium m tuberculosis (MTP)[37], Microcin M 492 from Klebsiellla pneumoniaae [38], and H Harpins from Xanthomonas X axonopodis [39] [ have beeen reported. Moreover, M recent studies haave indicated that amyloids are much moore widely disttributed amonng bacteria andd are useful foor biofilm form mation [35]. Here, H we descrribe some impoortant bacteriaal amyloids inn detail.

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Table 1. Functional amyloid in living systems Protein

Organism

Function

Ref.

Prokaryotic amyloid Bacterial amyloid Curli

Escherichia coli

MTP

Mycobacterium tuberculosis

Microcin

Klebsiella pneumoniae

Harpin

Chaplins

Erwinia amylovora, Pseudomonas syringae Streptomyces coleicolor

Fimbriae and bioflim formation formation that helps E.coli for host cell adhesion and colonization Amyloid of Pili helps in host adhesion and act as antigens for infection Toxic oligomers of Microcin causes pores on target bacterial cell membrane that is lost upon amyloid fibril formation

[26,40] [37] [38]

Amyloid of Harpins caused hypersensitive responses in plant cells

[39]

Cell surface proteins involved in formation of aerial hyphae and fruiting bodies

[48]

Eukaryotic amyloid

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Fungal amyloid Neurospora EAS crassa Schizophyllum SC3 commune HET-s

Podospora anserina

Sup35

Saccharomyces cerevisiae

Rnq1p

Saccharomyces cerevisiae

Ure2p

Saccharomyces cerevisiae

Amyloid of EAS helps in the formation of aerial structures as hyphae and spores. Amyloid of SC3 helps in the formation of aerial hyphae Amyloid of Het-s causes heterokaryon incompatibility that prevents the transfer of fungal cytoplasmic viruses Amyloid of Sup35 causes translational read through and results in altered proteome and phenotypic diversity in yeast ([PSI+]) Amyloid of Rnq1p induces the formation of [PSI+] phenotype in yeast Amyloid form of Ure2p causes the uptake of poor nitrogen source (e.g. ureidosuccinate) in presence of good nitrogen source

[84] [91] [102]

[29] [130] [110]

Mammalian amyloid Pmel

Mammals

Hormone

Mammals

Sequester the toxic intermediates during melanin synthesis Storage of hormones in secretory granules

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2.1. Curli Fibrils The curli fibrils are one of the major proteinaceous components of the extracellular matrix produced by E. coli during biofilm formation [40]. The major functions of curli fibril include binding to surface (or surface to the host cells) and to help the bacteria to survive and adapt in environment [26,36]. The biogenesis of curli fibers is mediated by two operons namely csgDEFG and csgBA, where the csgBA operon encodes the minor and major curli subunit proteins of CsgB and CsgA, respectively [26,40,41]. The stability and secretion of both CsgA and CsgB is dependent on the outer-membrane localized protein, CsgG [42]. The CsgE is suggested to play an important role in the stability of both CsgB and CsgA, while CsgF is known to be required for efficient curli biogenesis [40]. The CsgA and CsgB proteins are the main components of curli fibers. Both CsgA and CsgB has a domain composed of five glutamine-asparagine rich oligopeptide repeats of approximately 20 amino acids long [43]. Purified CsgA protein is capable to self-assemble into amyloid fibers in absence of CsgB protein [40]. However, in order to form CsgA amyloid, CsgB is required in vivo [44]. Cells that do not express CsgB, but only secrete CsgA into extracellular environment were unable to produce curli fibrils [19,26]. Interesting to note that same cell does not require expressing both the CsgA and CsgB for curli biogenesis. The CsgA secreted from a csgB-lacking mutant could be polymerized by CsgB, which is produced on the surface of a csgA-lacking mutant during interbacterial complementation [19,40,44-46]. The ability of CsgB to convert CsgA into an insoluble fiber suggests that CsgB acts as a curli nucleator protein [44]. CsgA polymerizes by a nucleation dependent polymerization mechanism similar to disease related amyloid formation mechanism [43] (Fig 2A). Several recent reports suggested that E. coli operates an elegant strategy to control curli fibril biogenesis that begins only by CsgB secretion that acts as a template for amyloid assembly of newly secreted CsgA [26,36]. Preformed CsgA fibrils can acts as a seed for folding and assembly template for additional soluble CsgA monomers [47]. By regulating the expression and secretion of CsgA and CsgB, the bacterial cell ensures curli fiber biogenesis occurs according to their requirement. Moreover, bacteria use this unique strategy to shield the potential toxicity of protein assembly intermediates, by promoting mature amyloid fiber formation in their extracellular space.

2.2. Pili of Mycobacterium Tuberculosis Mycobacterium tuberculosis is a pathogenic bacterium, which causes tuberculosis in humans. The main characteristic features of this bacterium include complex cell wall, intracellular pathogenesis, slow growth and dormancy. An outer layer beyond the peptidoglycan cell wall composed of lipids (especially mycolic acid) is important in pathogenesis and host inflammatory response. One of the adherence factors of M.tuberculosis is pili [37]. The pili are hairlike appendages found on the surface of bacteria that help them for adhesion, colonization and infection of the host. The pili of M. tuberculosis are called MTP that helps the bacteria to adhere to surfaces of macrophages, and therefore has a direct role in the pathogenesis of the organism [37]. Recently, it was reported that the pili of this bacteria is morphologically similar to curli fibrils and suggested to possess an amyloid-like structure by CR binding [37]. However, detailed study needs to be done to confirm the amyloid nature of pili.

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2.3. Chaplin Fibrils The gram-positive bacteria Streptomyces coleicolor and other streptomycetes possess a complex morphological differentiation where mycelium grows into air and subsequently divides into spores [34,48]. The newly developed spores are dispersed to allow the expansion of a new colonizing mycelium. Streptomycetes such as S. coelicolor and other streptomycetes are pathogens of animals or plants. In S. coelicolor, SapB, a 18 amino acid peptide was first found to be involved in aerial hyphae development [49,50]. Subsequently, the rodlin proteins, encoded by rdlA and rdlB, were found to be required for formation of hydrophobic rodlets [51]. Rodlins are necessary for filament adhesion but not for erection of aerial mycelium. In 2003, two independent reports suggested the group of proteins called Chaplins to act collectively for the formation of aerial filaments [48,52]. A family of eight related genes was identified, which show increased expression during aerial development. All the Chaplin proteins contain a hydrophobic domain called Chaplin domain with 60–65% hydrophobic residues. Three of the Chaplins, ChpA–C, are proteins of 210–230 residues called "long Chaplins" and contain two chaplin-domains separated by a ∼35 amino acids stretch. The other group of five Chaplins, ChpD–H contain a single Chaplin domain with range from 50–63 residues. Interestingly, the three longer chaplin proteins, ChpA–C, appear to be the substrates for the cell-wall sorting enzyme sortase that is involved in incorporating proteins into the bacterial peptidoglycan [53]. ChpA–C are therefore covalently bound to the cell wall and may facilitate the subsequent attachment of the shorter Chaplin proteins. It was found that the short Chaplins could still attach to aerial filaments even in mutants lacking the longer Chaplins. Short Chaplins could be extracted from filament cell walls by the treatment of trifluoroacetic acid (TFA) and resulting monomer are shown to be unstructured. However, monomers were readily assembled at air-water interface into amyloid like fibrils, which is of 4-6 nm in diameters [48]. The EM and ThT study confirm the formation of amyloid fibrils by Chaplins. It was suggested that Chaplins spontaneously self-assemble into amphipathic monolayer of amyloid fibrils. The hydrophobic surface of amyloid fibrils mediates attachment to other hydrophobic surfaces, facilitates penetration of the liquid-air interface by lowering the surface tension. This allows bacteria for the formation of aerial hyphae. Chaplins assemble in solution when seeded with the aggregated protein suggesting that nucleation dependent polymerization mechanism responsible for fibril formation. Chaplin fibrils are organized in rodlets at the surface of aerial hyphae and on spores from S. coelicolor. Although the role of SapB and rodlins in aerial hyphae formation is not clear, it is probable that rodlin proteins may help to organize the Chaplin fibrils for rodlet formation [54]. It is quite clear that Chaplin assembly into amyloids is required phenomenon for the aerial hyphae formation in filamentous bacteria. However, it is not clear whether the Chaplins assemble on the cell surface or at the water-air interface after release from the cells. More studies are required to understand whether any nucleator protein exist for Chaplins to form amyloid as observed for curli fibrils.

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2.4. Microcin Amyloid

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Bacteria produce toxins called ‘bacteriocins’ to inhibit the growth of closely related bacterial species. They do so by forming ion channels in the cytoplasmic membrane, degrading DNA, blocking protein translation, or inhibiting peptidoglycan synthesis [55]. Microcin E492 (Mcc) is a bacteriocin naturally produced by Klebsiella pneumoniae, which is active against several strains of Enterobacteriaceae [56,57]. The mechanism of action of microcin is associated with the formation of pores in the cytoplasmic membrane of target cells and subsequent loss of membrane potential [58,59]. Mcc activity is the highest when cells are in exponential growth phase and the activity is lost after cells enter into the stationary phase [56,57,59]. Although Mcc activity is very different in growth and stationary phase, the Mcc production, accumulation and molecular weight of Mcc purified from either growth phases, are unaltered [60,61]. Therefore it was proposed that the disappearance of activity in the stationary phase could be due to change in Mcc conformation and/or oligomerization state. Soto and his coworkers have shown that purified active Mcc can convert into amyloid-like fibrils in vitro after incubation [38]. They found that assembly of Mcc into amyloid fibrils also occurs in vivo and results in loss of the antibacterial activity of Mcc. The formation of amyloid fibril in vivo was correlated with the drop in Mcc activity in the culture suggesting amyloid formation modulates the activity of Mcc in vivo [38]. It was suggested that oligomeric intermediate, precursor of fibrils are produced in high amounts in the exponential growth phase that are cytotoxic. However, when bacteria enter into stationary phase, pore forming oligomers of Mcc are converted to amyloid fibrils with reduced toxicity. This study clearly indicates that although protofibrillar pore-promoting assemblies act as cytotoxic forms, the mature amyloids acts as inert protein deposits of aggregated Mcc without cytotoxicity [38]. Interestingly, oligomeric Mcc also showed cytotoxicity, which in turn induced apoptotic cell death in human cell lines [62].

2.5. Harpins of Plant Pathogenic Bacteria The hypersensitive response (HR) is a defense mechanism, used by plants, to prevent the spread of infection by microbial pathogens [63,64]. The HR is characterized by the rapid death of cells in the local region surrounding an infection to restrict the growth and spread of pathogens to other parts of the plant. The cell death in hypersensitive response is similar like apoptosis in animal cells. Some gram-negative pathogenic bacteria in plant secretes a class of proteins called harpins through the type III protein secretion system that induces hypersensitive response in plants [39]. The biochemical mechanisms by which harpins cause plant cell death are unclear. In 2007, Oh et al identified the first Harpin produced by Xanthomonas axonopodis pv. glycines 8ra (HpaG), which can self assemble into amyloid-like fibers [39]. The study suggested that His6-HpaG formed biologically active spherical oligomers, protofibrils, and β-sheet-rich fibrils. The fibrillar form of His6-HpaG is suggested to be an amyloid, based on positive staining with CR, increased protease resistance, and βsheet fibril structure. Further biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from α-helix to β-sheet-rich amyloid fibrils is important for the biological activity of the protein [39]. Accordingly, mutated harpins that unable to self-

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assemble into amyloid structures do not show the HR response in plant suggested that amyloid form of HpaG might be performing the HR activity [39]. To determine whether fibril formation is a common feature of harpins, other harpins such as XopA from X. campestris pv. vesicatoria, HrpN from E.amylovora and HrpZ from P.syringae pv. syringae have also been studied under plant apoplast-like conditions. In line of the previous observations that XopA, which is HpaG homolog but lack the ability to induce HR, also unable to form fibrillar structure. However, the gain-of-function mutant His6-XopA (F48L/M52L) was able to form curvilinear fibrils and protofibrils, suggesting a positive correlation between fibrillogenesis and HR activity. Moreover, HrpN and HrpZ also exhibited fibrillar structures, suggesting that amyloidogenesis is a common feature of harpins [39]. Therefore, the harpins are examples of functional amyloids, which are designed to be lethal.

2.6. Endospore

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Bacterial species such as Bacillus and Clostridium when exposed to environment with limited nutrients initiate the formation of endospore⎯ a unique structure that is highly resistant to heat, radiation, pH extremes, and toxic chemicals [65,66]. Morphologic analysis of the spore coat by atomic force microscopy has shown that it is principally composed of fibrillar structures with morphology similar with amyloids [67]. The inner and outer spore coats are composed of several proteins. However, the proteins involved in assembly and formation of amyloid fibrils are less understood. Further biochemical studies are required to characterize the proteins that form the spore coat, and to elucidate the amyloidogenecity of the protein.

2.7. Biofilms Biofilms are extracellular and structured communities produced by microorganisms for protection from environmental hazards [68,69]. These biofilms hold and/or embed the cells together and helps cells to colonize and attach to the surface. Although the specific composition of the extracellular matrix of biofilms varies from species to species, the main components include polysaccharides, proteins, and nucleic acids [70]. The most important groups of biofilm-associated proteins are those polymerize into fibers known as bacterial pili or fimbriae [26,35]. In E. coli, curli fibrils have been shown to be an integral part of this bacteria biofilm. As discussed above, Curli subunits assemble into amyloid fibrils in E. coli for helping them to colonize and for surface attachment [40]. The amyloid fibrils has been shown to be the part of extracellular matrix of biofilms in numerous microorganism including harpins of X.campestris and P.syringae, pili from M.tuberculosis, chaplins from S.coelicolor and hydrophobins from fungi. The gram-positive bacterium B.subtilis form endospores of matrix-encased biofilms, whose organization is largely dependent upon the presence of the extracellular matrix composed of an exopolysaccharide and the protein TasA [70]. It was suggested that TasA forms amyloid fibers that is essential for the integrity of the extracellular matrix and the biofilms. The purified TasA from B.subtilis have showed amyloid

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characteristics, which was functional in that it was able to restore wild-type biofilm forming ability to a mutant lacking TasA [70].

2.8. Bacterial Inclusion Bodies Contain Amyloid Like Structure

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E.coli often produce electron dense protein mass covered by membrane, called inclusion bodies (IB), during over expression of heterologous proteins [71-73]. The protein mass inside the IB is probably due to the amorphous aggregation of newly synthesized overexpressed protein. IB were often classified as amorphous, electron dense aggregates that lack any organized structure. However, several studies have suggested that IB might contain cross-βsheet rich amyloid structure reminiscent of amyloid fibrils [74-76]. IB can bind to amyloid specific dyes such as ThT, CR, could possess a seeding capacity as of amyloid fibrils, contain protein mass of β-sheet rich structure and display aggregation propensities strongly affected by mutations [33]. Recent study by Wang et al. using H/D exchange coupled with high resolution NMR technique, X-ray diffraction, ThT binding and CR binding have suggested that IB are indeed composed of cross-β-sheet rich amyloid. The amyloid gatekeeper mutation by arginine residue in the amyloidogenic region of overexpressed protein disrupted the formation of inclusion bodies and increased the protein production in soluble cytoplasmic pool [33]. Moreover, it was shown that IB of Het-s isolated from E. coli has the capacity to be infectious as like amyloid fibrils of Het-s [77]. This study clearly demonstrated that IB possesses amyloid-like ordered structure. All these studies supported the idea that during overexpression of protein, E. coli might intentionally store high concentrations of protein in amyloid-like structure inside the inclusion bodies for efficient storage of the protein and to protect themselves from the cytotoxic effect of protein oligomers.

3. Fungal Amyloid Fungi are eukaryotic organisms that include bread moulds, yeast and mushrooms. Most of these organisms exist as filamentous form, but unicellular forms such as yeast are also present. It has been suggested that amyloid fibrils are widespread in fungus either providing support for their hyphae/spore formation [19,20,34] or provide the selective advantage growth as by fungal prions [28,29,78,79]. Amyloid fibrils also provide support to fungi for their colonization as well as attachment to the host. Some of the most important fungal amyloids are described in the following sections.

3.1. Hydrophobins Like Chaplins in bacteria, fungi use a class of proteins called hydrophobins, which are small-secreted proteins that provide broad ranges of functions in fungal growth and development including the formation of hydrophobic aerial structures like aerial hyphae, spores and fruiting bodies [80-82]. Hydrophobins also allow fungi to escape their aqueous environment by providing enough hydrophobicity to fungal surfaces in contact with air and

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help fungi for the attachment of hyphae to hydrophobic surfaces [80]. The attachment to the hydrophobic surfaces is important for pathogenic fungi for their penetration and infection of the host. Based on their hydropathy patterns and solubility characteristics, two classes of hydrophobins exist namely class I and class II [82-84]. Class I hydrophobins are made up of cylindrical rodlets and are highly hydrophobic with less wettability, whereas the class II hydrophobins does not form rodlets [84]. The function of hydrophobins in fungal survival and their pathogenicity is based on the property of hydrophobins to self-assemble at a hydrophilic/hydrophobic interface into an amphipathic monolayer [80-82,85,86]. Hydrophobins are small proteins of around 100 amino acids, characterized by eight cysteine residues [86]. Self-assembly of hydrophobins results in an amphipathic monolayer, where the hydrophilic side of the amphipathic monolayer orients and attached to the cell wall and the hydrophobic side is exposed to the hydrophobic environment. This assists aerial hyphae and spores to become hydrophobic and reduces the water surface tension that eliminates physical barrier, which allows hyphae to grow into air. 3.1.1. SC3 Hydrophobin The amino acid sequences of hydrophobins are diverse but all contain eight cysteine residues that can form four-disulfide bond to stabilize hydrophobin monomeric fold [83,87]. SC3 from Schizophyllum commune is one of the well studied class I hydrophobins [81]. SC3 is a 112 amino acid protein of molecular weight 14 kDa. It self-assembles at the hydrophilichydrophobic interfaces into an amphipathic protein fibrils called rodlets. The rodlets of the class I hydrophobins of SC3 are amyloids in nature as they consist of two tracks of 2–3 protofilaments with a diameter of about 2.5 nm each with high degree of β-sheet structure. They can bind to amyloid specific dyes such as ThT and CR [81]. The disaggregated and monomeric SC3 can also polymerize in to rodlets structure with amyloid fibril like properties [81,85,86]. By reducing the disulphide bridges and/or blocking the sulfhydryl group with iodoacetamide, the protein spontaneously assembled in water [88]. The aggregated structure was indistinguishable from that of native SC3 assembled at the water-air interface. Several studies have suggested that hydrophobin self-assembly proceeds through complex mechanism of conformational transition, where water-soluble form proceeds via an intermediate of helical conformation, which is ultimately transformed into predominate β-sheet structure [89,90]. It was recently shown that SC3 can self assemble themselves to rodlets when the hydrophobin concentration and the time of incubation are high. A recent study has suggested that certain cell wall polysaccharides like schizophyllan and β-(1-3)–glucan can play an important role in promoting the formation of rodlets [91]. In contrast to class I hydrophobin, Class II hydrophobins can also assemble at hydrophilic-hydrophobic surfaces, but they do not form amyloid fibrils. The amphipathic structure of hydrophobin II is less stable and that could explain their inability to form rodlets. 3.1.2. EAS of Neurospora Crassa EAS are hydrophobins present in Neurospora crassa, which offer protection to aerial structures like spores and fruiting bodies [92,93]. They form an amphipathic monolayer over the aerial spores and hyphae, where the hydrophilic surface face in side and the hydrophobic surface are present outside, which gives water repellency to the spores or hyphae. It was shown recently that the aggregated EAS are amyloid-like fibrils because it stained with CR

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and shows green yellow birefringence, under cross-polarized microscopy [84,94]. The disaggregated and soluble EAS (‘easily wettable’) protein also could form polymeric structure on hydrophobic surface that can bind CR and produce birefringence when analyzed under cross-polarized light [94]. The dimensions of the fibrils are 10 nm in diameter, comparable to other amyloid structures and contain high β-sheet observed in CD spectra [94].

3.2. Adhesins of Yeast Adhesins of various yeast species that is responsible for cellular aggregation showed amyloid-like nature in vivo [95]. It was shown that the amyloidogenic sequences of the adhesins could form amyloid in vitro at concentrations very much lower than their actual concentrations in the cell. This implies adhesin proteins have an intrinsic property of amyloid formation and is an important component of cellular aggregation [95].

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4. Fungal Prions Prions are infectious proteins with amyloid fold that cause many genetically transmittable and sporadic diseases like Creutzfeldt-Jakob disease (CJD) and kuru in human, chronic wasting disease (CWD) in deer, scrapie in sheep, and bovine spongiform encephalopathy (also called ‘mad cow’ disease) in cow [96]. Fungi also have prions that are structurally similar and infectious as mammalian prions but unlike mammalian prions, they do not cause cell death, instead provide survival advantage to the host [28,29,79,97]. The term prion was coined by SB Prusiner to describe these infectious agents involved in Kuru disease [96]. The fungal prions are widely used as the model to study the neurodegenerative diseases caused by the mammalian prions. Fungal prions are cytoplasmic elements that show non-mendelian inheritance [78]. They are transferred from mother to daughter during budding and also exchanged between partners during mating. Thus due to prions, the cells with identical genotype can exist as different phenotypes and these traits are heritable.

4.1. Het-s amyloid in Podospora anserine In filamentous fungi Podospora anserina, an event of vegetative cell fusions occurs between different cells [98,99]. During cell fusion, mycelia of this fungus can fuse resulting cytoplasmic mixing and occasional mixing of nuclei between the cells. In this filamentous fungi, a process called ‘heterokaryon incompatibility’ induces the death of hyphae, when two strains of different genetic background fuse together [99-101]. The het locus controls the viability of the fused fungi, where heterokaryons with different het locus are destroyed. For the fused heterokaryon to survive, it requires genetic identity of the het genes [100]. The het locus possess two alleles namely het-s and het-S corresponding to two proteins of Het-s and Het-S, respectively. The Het-S is soluble protein whereas Het-s has the ability to convert to an aggregated form of prion state. The het-s allele encodes a protein with two different conformations: HET-s behaves as a prion and HET-s* does not. The het-S allele encodes the

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HET-S protein that is incompatible with [Het-s] prion strains but is compatible with neutral [Het-s*] strains. When fusion between two cells where one containing het-S and another containing het-s occurs, the aggregated Het-s interacts with soluble Het-S that induces the incompatibility reaction and cell death [102]. Although the exact mechanism of cell death is not clear yet, it was suggested that after aggregation, Het-s could form amyloid that have prion like properties and is infectious. Maddelein et al. showed that that the heterokaryon incompatibility reaction could also occur when cells bearing Het-S are transformed with amyloid fiber from recombinant Het-s [30]. The structural and biochemical analyses of recombinant HET-s protein have indicated that in vitro aggregates possess amyloid-like properties as studied by EM and high β-sheet content [30]. These aggregates can seed the soluble form of the protein and greatly accelerate the aggregation of soluble protein suggesting nucleation dependent polymerization of amyloid aggregation. It was suggested that the soluble form of the HET-s protein has a N-terminal (1-240) folded domain (helical) and a C-terminal flexible domain [103]. Upon HET-s aggregation, the C-terminal part (218-289) forms an amyloid core rich in β-sheet content and resistant to proteolysis degradation. In vivo expression of the C-terminal domain fused with GFP is sufficient for [Het-s] infection and propagation [103]. Riek and coworkers determined the structural organization of the prion domain of HET-s (218-289) in its amyloid form using quenched hydrogen-deuterium exchange measured by two dimensional solution NMR [104]. Four segments were displayed slow exchange rates and therefore considered to be involved in intermolecular hydrogen bonds. The solid-state NMR measurements were performed to establish the high-resolution structure of this amyloid fold. All these studies have suggested four β-strands comprising residues 226-234 (β1), 237-245 (β2), 262-270 (β3) and 273-282 (β4) are linked by two short loops and by an unstructured, 15-residue-long segment between β2 and β3 comprises the Het-s(218-289) fold. Alteration of the structure by the introduction of the β-sheet breaking Pro residue at β-strand locations resulted in a loss of infectivity in the fungus [104] indicating that the structure of the amyloid responsible for infectious characteristics of HET-s prion [104].

4.2. Yeast Prions All the yeast prions identified till now are from Saccaromyces cerevisiae [28,78,105]. [URE3] and [PSI] were initially identified through genetic screens used to identify gene mutants in nitrogen metabolism and translational suppression, respectively. Later [PSI+] inducing [PIN+] phenotype was discovered as a new prion in yeast. 4.2.1. Amyloid of Ure2p in [URE3] In presence of rich nitrogen source such as ammonia or glutamine, yeast turns off their synthesis of certain enzymes and transporters necessary for utilizing poor nitrogen sources, such as proline or allantoate [106,107]. Ure2p regulates this “nitrogen catabolite repression” by binding the transcription factors such as Gln3p in the cytoplasm, preventing their entry into the nucleus and therefore preventing transcription of many genes, including DAL5 [106,108,109]. The ure2 mutant can use ureidosuccinate in spite of the presence of ammonia [110]. Therefore, the cells without active Ure2p, either due to deletion of the URE2 gene or

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due to [URE3] prion formation utilized poor nitrogen source even in presence of good nitrogen source [110]. Several studies showed that amyloid formation by Ure2p is the basis for [URE3] phenotype [111-113]. Ure2p fibrils have been shown in vivo in [URE3]containing cells [114]. The GFP fused form of Ure2p can aggregate in [URE3] cells, but not in wild-type cells [112]. Ure2p protein extracted from [URE3] strains has shown partially resistant to proteases [112]. Ure2p can form amyloid-like fibrils in vitro, which have similar protease resistance as that of extracts from [URE3] strains [113,115]. Ure2p fibrils bind the dye CR and produce characteristic yellow-green birefringence under cross-polarized light [111]. Ure2p consists of 1-89 amino acid residues N-terminal prion domain, which is flexible and contains unusually high glutamine (Q) and asparagine (N) contents (∼46%). The Cterminal functional domain consists of amino acids 90-354 is folded and sufficient for nitrogen regulation activity [113]. It was suggested that 1-65 amino acids of the Ure2p prion domain is sufficient for prion induction and propagation, and comprise the protease-resistant core in the amyloid form of Ure2p [111]. Overexpression of Ure2p N-terminal prion domain (1-65) in wild type cells is sufficient to induce the appearance of [URE3] phenotype, whereas the expression of C-terminal region in yeast cells lacking the ure2 gene restores the function of the gene [113]. Although the mechanism of [URE3] phenotype due to amyloid conversion of Ure2p is well understood, it might be possible that conformational transition and amyloid fibrils formation by N-terminal domain could incorporate the C-terminal domain into fibrils or the assembly of Ure2p into fibrils may mask the C-terminal region of Ure2p that is necessary to interact with Gln3p. However, either of the mechanisms could lead to the loss of function of Ure2p [116]. 4.2.2. Amyloid of Sup35 in [PSI+] The Sup35 protein is an essential component of the translation termination machinery of the yeast [117]. Normally, Sup35 recognizes stop codons and terminates protein synthesis. In prion phenotype, [PSI+] cells, wt Sup35p is self-assembled to amyloid aggregates, which is therefore unable to participate in translational termination [118] (Figure 3). This results in translational read-through at the stop codon and C-terminally elongated proteins are formed. The [PSI+] phenotype is also propagated into the daughter cells as cells divide and therefore fulfilling the prion phenomena [119]. Lindquist and coworkers demonstrated that the [PSI+] prion is advantageous under several growth conditions and may provide an alternative mechanism for phenotypic plasticity during certain environmental condition by altering the yeast proteome [29] (Figure 3). By tagging GFP along with Sup35p, it was found that the aggregation of this protein caused [PSI+] phenotype [118]. When sup35 changes into prion form, it become infectious because the aggregated insoluble form can convert soluble sup35 into insoluble type and that is also inheritable from mother to daughter during cell division [97]. It was also shown that this prion could be cured by the overexpression of Hsp104 [118,120]. This chaperone protein also helps in the proper segregation of these prion elements to the daughter cells. Overproduction of Sup35p increases the frequency of [PSI+] phenotype de novo [121,122]. The Sup35 contain a N-terminal prion domain (N) and C-terminal functional domain (C) [123,124] and between these two domains there is a charged and conserved middle domain (M) that helps Sup35 for its solubility and works as a switch from nonprion to prion state [125]. The N domain is essential for converting Sup35 to the prion state in vivo [122] and helps to convert soluble protein into amyloid fibril in vitro [126]. The

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NM domain off Sup35p is noot essential foor the translatiional role of the N t protein buut can form am myloid in vitrro by nucleatioon dependent polymerizatioon manner and sufficient too confer all asspects of prio on behavior [778,127,128] of full-length protein. p The recent r structure-function reelationship stu udy also clearrly indicates that t amyloid fibril structurre is necessaryy for prion prroperty of thee protein [1288,129]. Furtheermore, full-leength Sup35p can form am myloid, and thhis process is specifically seeded s by extrracts of [PSI+ +] cells, and not n by extractts of [PSI-] ceells [126].

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Fiigure 3. The sch hematic represenntration of Yeastt Sup35p aggregaation and PSI+ phenotype. p Sup335p acts as a traanslation termin nation factor and it recognizes stoop codons and teerminates proteinn synthesis in yeeast. In prion phhenotype [PSI+],, the Sup35p is self-assembled s too amyloid aggreggates and therefo fore is unable to participate p inn translational terrmination event.. This causes trannslational read thhrough and leadds to altered proteeome and phhenotypic diverssity in yeast.

4.2.3. Amylo oid of Rnq1 in [PIN+] The prion [PIN+] of yeasst causes the induction i of [P PSI+] prion annd hence its name stands ng’ [130]. Thee gene responnsible for this prion p encodess for the proteiin Rnq 1, a foor ‘PSI inducin prrotein that is rich r in asparaagine and glutaamine. Rnq1 was identifiedd as a result of o search to obbtain proteins with N/Q-rich domains forr other possibble prions in yeast y [130]. It is believed thhat Rnq1 indu uces [PSI+] acttivity by cross seeding to sup35 s [131,1332]. To confirm m its prion foorming ability y, the prion domain of Rnq1p was attacheed to C terminnal domain of sup 35 and it was found th hat the resultinng strain posseess the [PSI+] phenotype [130]. The overrproduction off any N/Q-rrich protein domain also could inducce [PIN+]-likke action [1331,132] as deemonstrated th hat poly Q-conntaining domaain of huntingtin promote thhe de novo connversion of Suup35 into [PS SI+]. In contrast nonamyloiddogenic variannts of huntinggtin and amylooid form of trransthyretin an nd α-synucleinn do not cause the [PSI+] [132]. [ Howeveer [PIN+] provved to be a prrion due to thee fact that proppagation of [P PIN+] requires the Rnq1 genne [133]. Overrproduction + off Rnq1 increases the frequeency of [PIN ], ] and aggregaation of Rnq1 correlated perrfectly with thhe de novo fo ormation of [P PIN+] [130,1331,133,134]. In I contrast too other two prions p in S. + ceerevisae wherre the inductioon of prion is due to the loss of function, [PIN ] is duee to gain of fuunction by forrmation of prioon of Rnq1. Itt was also sugggested that thhe prion domaain of Rnq1 (R RnqPD) polym merizes more readily r in vitroo than the full--length proteinn [135]. The aggregation a off Rnq1 is a nu ucleation depeendent processs, where aggrregation kinettics could be accelerated a

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by the addition of preformed aggregates as a seed. Electron microscopy data also suggested that it could form spherical oligomers, agglomerates and amyloid like fibrils that bind to ThT, contain β-sheet rich structure as evident from circular dichroism (CD), show a partial proteinase K resistance [130,134]. These studies indicate that the Rnq1p aggregates are amyloid fibrils. These in vitro amyloid fibrils by recombinant Rnq1p are infectious for yeast [134]. The aggregation reaction of Rnq1 and Sup35 could also be cross-seeded by each other suggesting the possible role of Rnq1 in [PSI] phenotype [135] although Rnq1 and Sup35 formed separate fibrils in vivo [136].

5. Mammalian Functional Amyloid Functional amyloids are widespread in prokaryotes and lower eukaryotes, where they perform the directed function in favor of the organism. The era of functional amyloid in mammals has started after discovery of Pmel amyloid in melanosome by Jeff Kelly and his coworkers in 2005 [31]. Recently it was also shown that secretory granules of pituitary contain amyloid like structures to store the protein/peptide hormones for long time [32].

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5.1. Biogenesis of Mammalian Melanosome Melanosomes are lysosome-related mammalian cellular organelles, which generates and stores the melanin pigment in specialized cells including melanocytes and retinal pigment epithelium cells that reside in the skin and eyes respectively [137,138]. Melanosome biogenesis occurs through four distinct sequential steps [139-141]. In stage I, vesicular structures lacking melanin called premelanosomes are endosomally formed. Subsequent maturation of premelanosomes with the development of striated appearance due to the intralumenal protein fibers takes place in Stage II. The synthesis and deposition of melanin onto the protein fibers occurs in stage III melanosomes, which possess the characteristic dark and thick striations. In stage IV, the accumulation of melanin occurs that masks all the intralumenal structures. It has been suggested that melanosome maturation depends on the presence of specific cargo proteins that are delivered to the appropriate-stage of melanosome through highly regulated sorting mechanisms. 5.1.1. Pmel Amyloid and Melanin Synthesis The striations that are formed during the maturation of melanosome are proteinaceous in nature, and are made up of a fragment of Pmel17, a membrane protein of melanosome [142]. A correlation was observed between the expression of Pmel17 and melanin synthesis and therefore it was suggested that Pmel17 plays an important role in melanin synthesis [143]. Pmel17 is associated with intralumenal vesicles in early endosomes as well as premelanosomes and becomes significantly enriched in stage II premelanosomes in association with striations development [137,138,141,144]. The importance of Pmel17 in melanosome biosynthesis is further supported by the fact that exogenous expression of Pmel17 in non-pigmented cells resulted in the formation of Pmel17-containing striations within multivesicular bodies [145]. Pmel17 is a type 1 transmembrane protein with a large

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lumenal domain and a single transmembrane and short cytoplasmic tail [146]. The processing of Pmel 17 is mediated by furin cleavage or cleavage by proprotein convertase enzyme in post-Golgi compartment that convert Pmel17 into two fragments; 80 kDa lumenal N-terminal fragment, Mα and a 28 kDa membrane associated C-terminal fragment, Mβ [142,145]. The processing of Pmel17 is required for striation formation was evident when striations was blocked either by inhibition of furin cleavage or by exogenous expression of a proprotein convertase inhibitor. In addition to enzymatic cleavage of Pmel17, it is essential that Mα should be dissociated from Mβ by breaking of disulfide bond between Mα and Mβ. After releasing from Mβ, Mα probably undergoes a conformational transition and self-association into fibrils, which constitute the melanosomal fibers. The self-assembly of Mα could also be accelerated by low lumenal pH of the maturing melanosome. The Mα fibrils are then sequestered by the intralumenal vesicles (ILVs) of multivesicular bodies or early endosomes, which becomes premelanosomes and they are recruited for the formation of striations in the stage II melanosomes [145] (Figure 4A). In 2006, Jeff Kelly and his coworkers reported that isolated Mα fibers from melanosome of bovine eyes have amyloid like properties such as the ability to bind CR and Thioflavin S [31]. To further support their hypothesis, a 442-residue lumenal fragment of Pmel17 (recombinant Mα; rMα) was expressed, which form amyloid fibrils almost instantly. The formation of the amyloid by recombinant Mα was confirmed by the high binding to ThT and CR, formation of β-sheet structure by far-UV CD and FTIR. The cross-β-sheet structure of amyloid was further confirmed by X-ray powder diffraction study, which showed two reflections 4.6 Å and 10 Å [31]. The instantaneous amyloid formation suggests that Mα amyloid fibrils formation is evolutionary optimized for performing native function. The role of Pmel17 amyloids in melanin biosynthesis was studied utilizing rMα amyloid fibrils. Melanin synthesis includes indole 5,6-quinone (DHQ) and other intermediates that polymerize on the Mα-fibril template in the melanosome. An in vitro melanin polymerization assay was initiated where tyrosinase, 3-4 dihydroxyphenyalaine (DOPA) and rMα amyloid were added together to reconstitute the in vivo melanin synthesis [31] (Fig 4A). A time course of melanin synthesis in vitro revealed that rMα fibrils highly accelerated the polymerization of melanin precursor to melanin polymers. It was also shown that the amyloids of Aβ and αsynuclein were also able to catalyze melanin formation in a rate comparable to rMα, suggesting that the cross-β-sheet motif of amyloid acts as a template for the polymerization of melanin precursors [31]. The data suggests that amyloid templates of Pmel17 accelerate the polymerization of reactive melanin precursor into melanin, thus reducing toxicity associated with melanin synthesis. Pmel amyloid could also play a role in alleviating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. To understand the Pmel processing, aggregation and amyloid formation in melanosome biogenesis, Hurbian et al. recently performed high-pressure freezing (HPF) and electron tomography (ET) using MNT-1 melanoma cells where they were able to observe the internal organization of early melanosomal intermediates at high resolution [147]. The data suggested that amyloid fibrils are fully formed in stage II premelanosomes where they organize into sheet-like arrays. Subsequently, melanin deposits occurs into these fibrillar sheets at the stage III premelanosome [147].

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Fiigure 4. Function nal amyloid in mammals. m (A) Scchematic represeentation of Pmel amyloid formattion and m melanin polymerization in melanoosome biogenesiis: Pmel17 is a trransmembrane protein p and after processing byy furin cleavage or cleavage by proprotein p conveertase, it producees two fragmentts of Mα and a membrane m asssociated C-term minal fragment, Mβ. M After breakaage of disulfide bond, b Mα gets separated s from Mβ M and suubsequently confformational transition of Mα leaads to aggregatioon and amyloid formation f in prremelanosmone stage II. The higghly reactive meelanin precursor subsequently binnds to Mα fibrilss and reesulting melanin polymerization in stage III. In stage s IV, the accuumulation of meelanin masks all inntralumenal struccture (not shownn here). Letters of o a, b, c and d reepresent active melanin m precursoor, melanin prrecursor, Pmel am myloid and melaanin polymer, reespectively. (B) Schematic S representation showinng amyloid ass a natural storag ge in secretory grranules of proteiin/peptide hormoone: Protein/pepptide hormones aggregate a seelectively into am myloids, and subbsequently sequeestered by membbrane to form seccretory granules. The m monomeric formss of hormones arre then released to t the extracellullar space to perfform their functioons, upon exxternal stimuli when w needed. Thee parallel arrays of lines represennt β-sheet structture of amyloid aggregates a in seecretory granule (SG).

5.2. Amyloid d in Secreto ory Granule es Biogenes sis There are mainly two tyypes of protein secretion in secretory cells of eukaryyotes [148w proteins are transpoorted immediiately after 1551]. One is "constitutive secretion" where syynthesis throu ugh ER-Golgii route to thee extracellularr space. In thhe constitutivee secretion, trransport and fu usion of the vesicles v to the plasma membbrane for releaasing the vesiicle content dooes not requirre any external stimulus. Therefore, T in the constitutivve secretion, the rate of prrotein synthessis is similar to the rate off protein secrretion in the extracellular e s space. It is beelieved that constitutive secretion is the default pathw way for proteiin secretion inn secretory ceells. Many ceells such as liiver and musccle cells use this t constitutiive route for the t protein seecretion. The other route foor protein secrretion is calledd "regulated secretion" that is used by seeveral cells in ncluding endoccrine, neuroenndocrine and mast m cells, whhere the cells are able to sttore secretory y proteins/pepptides for lonng periods inn a highly concentrated c form. The cooncentrated fo orm of proteinn/peptides aree membrane-enclosed and appear a as elecctron-dense

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cores in the cytoplasm of the cell under electron microscope. These electron dense cores are termed “secretory granules” [149,150,152,153]. The electron dense core of secretory granules is due to the condensation of granules contents, mainly composed of stable, intermolecularly aggregated proteins/peptides. Unlike the constitutive secretion, cells retain the secretory granules in regulated secretion until they receive signals to release their contents into the extracellular space. It is believed that reversible aggregation is the necessary step for condensing and sorting the secretory granule proteins for secretory granules biogenesis [150157]. Many secretory proteins have shown to form intermolecular aggregates in vitro at granules relevant conditions, and aggregates of proteins were also observed in vivo, suggesting that aggregation is the underlying mechanism for protein sorting into regulated secretory pathway. Although the aggregation of regulated secretory protein is known for long time, it is not however clear yet whether the aggregated and concentrated proteins inside the granules are amorphous or contain any specific structure. Several previous studies have indicated that secretory granules contain stable structure [158-161]. However, the structural properties were not completely understood. It was recently proposed that amyloid-like structure formation could be the mechanism for aggregation and sorting for pituitary secretory granules [32] (Fig 4B). More than 40 peptide/protein hormones were chosen for in vitro amyloid formation study, wherein more than 30 hormones formed amyloid in presence and/or absence of heparin, a representative glycosaminoglycan [32]. Most of the remaining nonamyloid forming peptide/protein under study formed amyloid in presence of amyloid forming partner peptide or glycosaminoglycans other than heparin. To establish whether secretory granules of pituitary also contain an amyloid-like structure, the secretory granules were isolated from AtT20 cells and rat pituitary. The secretory granules from both cell lines and rat pituitary appeared to bind amyloid specific dyes such as ThT and CR, and to amyloid specific antibodies. The CR birefringence study and X-ray powder diffraction study with isolated granules further supported the presence of cross-β-sheet structure in the secretory granules [32]. The immunofluorescence study of mouse pituitary tissue utilizing Thio S and antibody against pituitary hormones further indicated that hormones are present inside the pituitary granules in amyloid like structure [32]. Interestingly, these hormone amyloids were found to be less cytotoxic than that of Aβ amyloid (associated with Alzheimer’s disease). The hormone amyloids were able to release monomeric hormone upon dilution, and these released monomers retained their activity and conformation [32].

6. Other Functional Amyloids Amyloids performing native functions of the host are also found in other members of the animal world like insects, mollusks etc. The strong and rigid silk (spider silk) of spider web was reported to show an amyloid-like cross-β-structure [162,163]. The amyloids of chorion are found in the eggshell of the silk moth, where it protects the developing embryos from environmental hazards [27]. The neuronal CPEB protein of the sea hare, Aplaysia californica (ApCPEB) that contains a Q/N rich domain can form amyloid state, which has similar characteristics yeast prions [164]. The aggregative amyloid state of CPEB is the functional, RNA-binding form of the protein. CPEBs are important for memory retention due to their ability to activate dormant messenger RNA transcripts near neuronal synapses [165].

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7. Functional vs Disease Amyloid Although amyloids are suggestive to be pathogenic species in several amyloid diseases, the growing discovery of “functional amyloids” in many organisms, including bacteria, fungi, and mammals (Table 1) suggest that amyloid fibrils are evolutionary optimized protein fold capable of performing native biological functions [18]. The discovery of many functional amyloids also provides a unique platform for understanding the amyloidogenesis process, which is essential for drug discovery against amyloid diseases [1]. Although both the disease and functional amyloids are formed via nucleation dependent polymerization reaction (Fig 2A) by producing cytotoxic oligomers, the mature fibrils of functional amyloids become less toxic or non-toxic compared to disease amyloid. Most of the functional amyloid as discussed in this chapter, aggregates into higher order fibers in vivo, which are non-toxic and perform native function of the host [18,20,31,32]. It is highly probable that irrespective of disease or function, the precursor oligomers of amyloid could be cytotoxic. This is further supported by the fact that oligomers from non-disease associated protein produce cytotoxicity [166]. Moreover, the aggregation intermediate of Mcc amyloid fibril is cytotoxic but not the mature amyloid fiber [38]. The lesser toxicity associated with mature fibrils suggests that amyloid fibrils might be a mechanism for detoxification. However, functional amyloid proteins could end up in forming disease amyloid or could accelerate disease amyloid formation, if it is unregulated. Such regulations require a functional protein homeostasis [167]. Both functional and disease amyloids could exist in the same organism even in same organ. If the protein homeostasis is altered under certain conditions such as diet, stress and age, functional protein aggregation may not be regulated anymore and disease-associated amyloid aggregation may occur. For example, amylin that is stored in secretory granules, forms amyloid fibrils in vitro, and is present in an amyloid state in the pancreas of type II diabetes patients. It is not clear yet if the aggregation causes the disease, or it is an indirect effect of the altered protein homeostasis. Furthermore, studies have suggested that injection of curli in the mouse model of AA amyloidosis, could accelerate the disease progression suggesting the clear notion that functional amyloid could lead to a danger by cross seeding to disease amyloid formation [168]. It is therefore necessary for the cell to evolve mechanisms to control and propagate functional amyloid formation so that the cytotoxicity of oligomers and amyloid fibrils (if any) are diminished. Different organisms have dealt with regulation of amyloid fibril formation in different way. For example, Curli fibril formation in E.coli is highly regulated and the assembly machinery ensures that the curli subunits interact at the correct time and location [26]. The maintenance of yeast prion are chaperones dependent. For example, heat shock protein 104 (HSP104) has shown to be required for prion propagation and solubilization of aggregated proteins [120]. In melanosome biosynthesis, a tight regulation of amyloid fibrils formation exist, where the full-length Pmel17 is non-amyloidogenic when it is freshly synthesized and trafficked to early melanosomes as a transmembrane protein [146]. However, after sequestered in the early melanosome compartment, Pmel17 release the amyloidogenic fragment, Mα, by proteolysis. Very fast kinetics of self-assembly of Mα fibrillation and its membrane encapsulation probably minimizes the toxicity [31]. Similarly, in secretory granule biogenesis, hormone amyloids may not be (very) toxic since they are stored inside the granules and the amyloid aggregation of hormones for secretory granule formation may be highly regulated that includes 1) processing of prohormones that aggregate more slowly than

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the hormone counterpart as shown for amylin [169]; 2) in the absence of prohormone processing, amyloid aggregation might be controlled by appropriate amyloid helper molecules as observed for prolactin, which only aggregates in presence of chondroitin sulfate A; 3) the decreasing pH gradient from ER to Golgi and Cis-Golgi to trans-Golgi and to secretory granules. Furthermore, the hormone amyloids are stored in an “inert” membrane container and the amyloid fibrils dissociate upon secretion [32]. It is also interesting that both disease and functional amyloid possess similar morphology and consists of cross-β-structure (Figure 2). However, amyloid fibrils associated with diseases are more toxic compared to functional ones suggesting that little quaternary structural differences might exist or higher order organizational difference of protofilaments in amyloid fibrils might account for different toxicity. For example, the lack of toxicity of HET-s fibrils could be due to their hydrophilic surface that may suppress non-specific hydrophobic interactions with other proteins or the membrane [2]. Alternatively, functional amyloid proteins may aggregate into highly specific amyloid fibrils without significant population conformational intermediates such as protofibrils and oligomers that are in equilibrium with amyloid fibrils. It is therefore necessary to determine the quaternary structure at atomic resolution of both functional and disease associated amyloid to compare any structural difference for the toxicity [2]. It is also important to note that amyloid inhibitors designed against amyloid diseases could interfere with the amyloid aggregation in functional amyloid. For example inhibitors drug against Alzheimer's could interfere with the melanosome biosynthesis. Therefore, amyloidogenesis inhibitors must be designed with sufficient specificity to avoid interfering with these native functions.

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8. Conclusion and Future Direction Although the association of the amyloids with many human diseases cannot be ignored even after suggestion that oligomers are most probable pathogenic species, the diverse list of functional amyloid with cross-β-sheet structure certainly suggest that amyloid is also evolutionarily conserved protein structure and are able to perform native biological function. The unique structure, morphology and stability against harsh physical and chemical conditions suggest that functional amyloid is more widespread in biological system. Functional amyloid appears to play an important role in mammalian melanosome biogenesis, secretory granules biogenesis, and plays a significant role in insect, bacterial, fungal and yeast function. In future, many more functional amyloids will be discovered and detailed functions of these existing amyloids will be uncovered. Understanding the functional amyloids certainly would inspire the researchers to develop novel bio-inspired functional materials, as many groups already have utilized amyloids as scaffold for development of bioactive materials [23,170,171], silver nano wires [21], substrate for neurite outgrowth, synapse formation, tissue repair and tissue engineering [22,23].

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Acknowledgment We acknowledge the IRCC (IITB) and CSIR (Grant no. 10CSIR 001) for financial support.

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[5] [6] [7]

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[8]

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[130] Sondheimer, N., and Lindquist, S. (2000). Rnq1: an epigenetic modifier of protein function in yeast. Mol Cell, 5, 163-172. [131] Derkatch, I.L., Bradley, M.E., Hong, J.Y., et al. (2001). Prions affect the appearance of other prions: the story of [PIN(+)]. Cell, 106, 171-182. [132] Derkatch, I.L., Uptain, S.M., Outeiro, T.F., et al. (2004). Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro. Proc Natl Acad Sci U S A, 101, 12934-12939. [133] Derkatch, I.L., Bradley, M.E., Masse, S.V., et al. (2000). Dependence and independence of [PSI(+)] and [PIN(+)]: a two-prion system in yeast? EMBO J, 19, 1942-1952. [134] Patel, B.K., and Liebman, S.W. (2007). "Prion-proof" for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+]. J Mol Biol, 365, 773-782. [135] Vitrenko, Y.A., Gracheva, E.O., Richmond, J.E., et al. (2007). Visualization of aggregation of the Rnq1 prion domain and cross-seeding interactions with Sup35NM. J Biol Chem, 282, 1779-1787. [136] Bagriantsev, S., and Liebman, S.W. (2004). Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast. J Biol Chem, 279, 51042-51048. [137] Marks, M.S., and Seabra, M.C. (2001). The melanosome: membrane dynamics in black and white. Nat Rev Mol Cell Biol, 2, 738-748. [138] Raposo, G., and Marks, M.S. (2007). Melanosomes--dark organelles enlighten endosomal membrane transport. Nat Rev Mol Cell Biol, 8, 786-797. [139] Dell'Angelica, E.C. (2003). Melanosome biogenesis: shedding light on the origin of an obscure organelle. Trends Cell Biol, 13, 503-506. [140] Seiji, M., Fitzpatrick, T.B., Simpson, R.T., et al. (1963). Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature, 197, 1082-1084. [141] Raposo, G., Tenza, D., Murphy, D.M., et al. (2001). Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J Cell Biol, 152, 809-824. [142] Berson, J.F., Theos, A.C., Harper, D.C., et al. (2003). Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis. J Cell Biol, 161, 521-533. [143] Kwon, B.S., Halaban, R., Kim, G.S., et al. (1987). A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutylmethyl xanthine. Mol Biol Med, 4, 339-355. [144] Raposo, G., Marks, M.S., and Cutler, D.F. (2007). Lysosome-related organelles: driving post-Golgi compartments into specialisation. Curr Opin Cell Biol, 19, 394-401. [145] Berson, J.F., Harper, D.C., Tenza, D., et al. (2001). Pmel17 initiates premelanosome morphogenesis within multivesicular bodies. Mol Biol Cell, 12, 3451-3464. [146] Harper, D.C., Theos, A.C., Herman, K.E., et al. (2008). Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of Pmel17 that have been proteolytically processed in endosomes. J Biol Chem, 283, 2307-2322. [147] Hurbain, I., Geerts, W.J., Boudier, T., et al. (2008). Electron tomography of early melanosomes: Implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci U S A,

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[148] Kelly, R.B. (1987). Protein transport. From organelle to organelle. Nature, 326, 14-15. [149] Kelly, R.B. (1985). Pathways of protein secretion in eukaryotes. Science, 230, 25-32. [150] Arvan, P., and Castle, D. (1998). Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J, 332 ( Pt 3), 593-610. [151] Arvan, P., and Castle, D. (1992). Protein sorting and secretion granule formation in regulated secretory cells. Trends Cell Biol, 2, 327-331. [152] Dannies, P.S. (2001). Concentrating hormones into secretory granules: layers of control. Mol Cell Endocrinol, 177, 87-93. [153] Dannies, P.S. (2002). Mechanisms for storage of prolactin and growth hormone in secretory granules. Mol Genet Metab, 76, 6-13. [154] Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science, 189, 347-358. [155] Dannies, P.S. (1999). Protein hormone storage in secretory granules: mechanisms for concentration and sorting. Endocr Rev, 20, 3-21. [156] Dannies, P. (2003). Manipulating the reversible aggregation of protein hormones in secretory granules: potential impact on biopharmaceutical development. BioDrugs, 17, 315-324. [157] Arvan, P., Zhang, B.Y., Feng, L., et al. (2002). Lumenal protein multimerization in the distal secretory pathway/secretory granules. Curr Opin Cell Biol, 14, 448-453. [158] Keeler, C., Hodsdon, M.E., and Dannies, P.S. (2004). Is there structural specificity in the reversible protein aggregates that are stored in secretory granules? J Mol Neurosci, 22, 43-49. [159] Miller, F., Harven, de E. and Palade, G. E. (1966). The Structure of Eosinophil Leukocyte Granules in Rodents and in Man. The Journal of Cell Biology, 31, 349-362. [160] Fedorko, M.E., and Hirsch, J.G. (1965). Crystalloid Structure in Granules of Guinea Pig Basophils and Human Mast Cells. The Journal of Cell Biology, 26, 973-976. [161] Greider, M.H., Howell, S.L., and Lacy, P.E. (1969). Isolation and properties of secretory granules from rat islets of Langerhans. II. Ultrastructure of the beta granule. J Cell Biol, 41, 162-166. [162] Kenney, J.M., Knight, D., Wise, M.J., et al. (2002). Amyloidogenic nature of spider silk. European Journal of Biochemistry, 269, 4159-4163. [163] Slotta, U., Hess, S., Spiess, K., et al. (2007). Spider silk and amyloid fibrils: a structural comparison. Macromol Biosci, 7, 183-188. [164] Si, K., Lindquist, S., and Kandel, E.R. (2003). A neuronal isoform of the aplysia CPEB has prion-like properties. Cell, 115, 879-891. [165] Si, K., Giustetto, M., Etkin, A., et al. (2003). A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia. Cell, 115, 893-904. [166] Baglioni, S., Casamenti, F., Bucciantini, M., et al. (2006). Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci, 26, 8160-8167. [167] Balch, W.E., Morimoto, R.I., Dillin, A., et al. (2008). Adapting proteostasis for disease intervention. Science, 319, 916-919. [168] Lundmark, K., Westermark, G.T., Olsen, A., et al. (2005). Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism. Proc Natl Acad Sci U S A, 102, 6098-6102.

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[169] Yonemoto, I.T., Kroon, G.J., Dyson, H.J., et al. (2008). Amylin proprotein processing generates progressively more amyloidogenic peptides that initially sample the helical state. Biochemistry, 47, 9900-9910. [170] MacPhee, C.E.W., D.N. (2004). Engineered and designed peptide-based fibrous biomaterials. Curr. Opin. Solid State Mat. Sci., 8, 141-149. [171] Scheibel, T. (2005). Protein fibers as performance proteins: new technologies and applications. Curr Opin Biotechnol, 16, 427-433.

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In: Protein Aggregation Editor: Douglas A. Stein, pp. 111-137

ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 4

Nucleation Mechanisms and Morphologies in Insulin Amyloid Fibril Formation Vito Foderà, Fabio Librizzi, Valeria Militello, Giovanna Navarra, Valeria Vetri and Maurizio Leone

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Dipartimento di Scienze Fisiche e Astronomiche, Università degli Studi di Palermo, Via Archirafi 36, 90123 Palermo, Italy

Abstract Aggregation processes, and in general the physical and chemical instability of proteins, are at the moment a major problem related to different scientific fields, spanning from biochemistry and biophysics to pharmaceutical and medical sciences. In fact, increased knowledge on protein aggregation may clarify different aspects related to several degenerative pathologies like Alzheimer's and Parkinson's diseases and type-II diabetes. In this chapter, we present and discuss our experimental results on thermally induced aggregation of a model system protein, the hormone insulin. This molecule is largely used in protein-based drugs and exhibits a great propensity to form amyloid aggregates with mechanisms similar to those of other disease-related proteins. Therefore, insights on insulin stability in adverse conditions as well as on mechanisms of fibril formation have a double relevance. Using the scientific approach developed by our group in the last few years, we analyze the fibrillation kinetics of insulin as a function of the initial protein concentration paying particularly attention on the balance between different nucleation-elongation mechanisms and their effects on the final fibril morphologies. Using the fluorescence properties of amyloid sensitive dye Thioflavin T (ThT), static/dinamic light scattering and atomic force microscopy (AFM), the main role of secondary nucleation in determining the well-known exponential time course has been revealed. Specifically, the role of the early stable fibril surfaces and their ability in catalyzing the fibrillation reaction has been considered. Moreover, the fine investigation of the early stages of the

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Vito Foderà, Fabio Librizzi, Valeria Militello et al. process shows a pronounced stochasticity in the first aggregation events leading to an overall spatial heterogeneity in the formation of the early stable amyloid fibrils. These evidences are discussed and lead us to suggest a picture of interconnected events taking place at different stages in the process.

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1. Introduction Protein aggregation can certainly be considered one of the most interesting and challenging topics in current research. It is concerned with a transition of a frustrated system like proteins from an initial (soluble precursors) to a final phase (insoluble aggregates) involving complex intra and intermolecular interactions, modulated by initial protein structure and physico-chemical properties of the environment. Several studies indicate that a general characteristic of aggregation processes is the multiple interaction and cross-feedback among different mechanisms occurring at different hierarchic levels, from single protein to oligomers to fibrils, and at different time scales, depending on external conditions. Among these mechanisms of utmost importance are protein conformational changes (possibly including oligomers formation), nucleation and growth of different kinds of aggregates (amorphous structures, fibrils, fibers, gels) and phase transitions of the solution like the liquid-liquid demixing [1-6]. Elucidation of the molecular mechanisms may also have great relevance in different medical fields [7-9]. In fact, several neurodegenerative pathologies have been shown to be strictly related to the presence of ordered protein aggregates in the injured tissues, their role in the diseases still being a matter of debate [10-14]. Moreover, aggregation processes are at the moment a major issue in the development of protein delivery systems. For this reason, a deeper understanding of molecular mechanisms at the basis of aggregate formation is also needed to improve purification, storage and delivery of protein-based drugs [7]. Ordered protein aggregates, referred to as amyloid fibrils, are elongated structures characterised by cross β-structures aligned perpendicularly to the fibril axis and the common highly organized hydrogen-bonded structure gives them a unique kinetic stability [15]. Noticeably, the formation of such species does not take place only in vivo and under specific conditions, fibril occurrence can be observed also in vitro by a number of different proteins [13, 14]. As a consequence, the capability of proteins to lose their native structure and selfassembly into amyloid fibrils seems to be a general property of any polypeptide chain [14]. For this reason, studying in vitro amyloid formation of model proteins allows us to shed new light on the involved mechanisms taking place in vivo. Amyloid aggregation processes are generally indicated to proceed through different steps within different temporal and length scales. Conformational changes are commonly accepted to be a main stage in the aggregation pathway [12, 16-19] in which partially destabilized and aggregation prone structures appear. In general, amyloid formation is described in terms of the so called “nucleation and elongation” model [20-23]. Destabilized structures are able to interact with each other and form a new high energy species called “nucleus”; protein molecules interact with such new species starting the elongation phase and leading to the formation of mature amyloid fibrils [22]. The elongation process may proceed via different mechanisms leading to specific temporal features for the fibril growth. As shown for several proteins [24-26], not only an end-to-end attachment of protein monomers to the nucleus

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(homogeneous nucleation), but also highly cooperative pathways may take place (secondary nucleation), involving the already formed fibrils as activators for new fiber filaments (branching, fragmentation, heterogeneous nucleation) [22]. This may results in a mixture of different morphologies As pointed out by Ferrone (1999), the kinetic features of both these growth processes can be mathematically rationalized, in terms of a quadratic and an exponential growth time dependence for the homogenous and secondary nucleation, respectively [22].

1.1. Nucleation Mechanisms in Protein Aggregation

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A main goal of a mathematical modeling of protein aggregation kinetics is to describe the time course of the reaction also predicting the dependence of the temporal features of the process on critical parameters such as temperature and protein concentration. To build up a model, a molecular mechanism needs to be assumed, so that the rate constants of the process together with their dependence on the parameters can be obtained. With the aim of supporting the description and discussion of the experimental results, in the following sections a short introduction of the mathematical approach to describe the nucleation processes will be provided. The description is mostly based on the concepts and the mathematical modeling developed by Ferrone and coworkers in the 80's, working on HbS polymerization. A more detailed and deep description of these results is elegantly presented by Ferrone in a complete and exhaustive review [22]. 1.1.1. Homogeneous and Secondary Nucleation Amyloidogenic proteins, like other fiber-forming proteins, have been suggested to selfassembly by a nucleated polymerization mechanism. In such a process two main steps can be identified. The initial step consists of a number of unfavourable equilibria that makes difficult the formation of a nucleus by subsequent monomers. Once oligomers of critical size are formed, another sequence of thermodynamically favourable reactions takes place, and attachment of a monomer to the nucleus is characterized by new kinetic parameters with a very high monomer-nucleus affinity that makes the reaction practically irreversible. The last step is known as the elongation mechanism and proceeds up to a complete depletion of the monomer population. Nucleated polymerization has several well-known features according to the classical model: 1) a critical concentration, below which fibrils cannot form, 2) a strong dependence of the fibril formation rate on concentration, which increases with the size of the nucleus [22, 27, 28]. The temporal behavior of the polymerization reaction can be predicted. If c* and J* are the concentration of nuclei and the rate of elongation of the nucleus, respectively, a differential equation for the time-dependence of polymer concentration (cp) can be written:

dc p dt

= J * ⋅c *

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Moreover, assuming that polymers can increase in mass only by attachment of monomers to their ends and each attachment proceeds with the same elongation constant J independent of the length of the polymer, a differential equation for the monomer that has gone into the polymer, [c0 - c(t)], can be established:

d [c0 − c(t )] = J ⋅ cp dt

(2)

Equations 1 and 2 together do not have an analytical solution and they can be only numerically integrated. Notwithstanding this, Ferrone developed a perturbative approach to simplify the system, consisting of the formal expansion of the equation about their initial values obtaining the analytical solution:

[c0 − c(t )] = A [1 − cos( Bt )]

(3)

where A and B are two parameters dependent on the rate constants and nucleus concentration. Further, a more intuitive approximation could also assume that near the beginning of the reaction all variables take their original values (J = J0; c = c0 and c* = c0*). Using such an assumption the system composed by equations 1 and 2 straightforwardly leads to the following solution:

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[c 0 − c (t )] =

1 J ⋅ J * ⋅ c* ⋅ t 2 2

(4)

that is an expansion about the initial value of the function in equation 1. It must be noted, however, that, because of the pertubative procedure employed, the expression in equation 3 is only a good approximation for the initial part of the kinetics. Both the functions in equations 3 and 4 are shown in Figure 1. The lag phase is almost absent and, as expected, the two solutions are superimposed over each other in the early stages. The hypothesis on the elongation mechanism leading to equations 1 and 2 includes only elongation through simple addition of monomers or, better, the so called primary or homogeneous nucleation. However, the mechanism described above is not of general character. Together with the homogeneous nucleation other reactions can take place, which are also dependent on the ongoing polymerization. These additional processes are commonly indicated as secondary nucleation and they can occur with different molecular mechanisms. Generally, three possibilities are indicated: fragmentation, branching and nucleation on the already formed fibrils, the latter also called heterogeneous nucleation. Fragmentation is mainly based on the breaking of a part of the polymer chain. Such part presents additional ends to start a new polymer growth with a final result of incrementing the rate constant of the overall polymerization. Branching includes the addition of a monomer, or generally of a subunit, on a specific site already present in the forming polymer. Finally, the heterogeneous nucleation is effective when a minimum length of the polymeric chain is provided. Then, the so called heterogeneous nucleus is formed and a new elongation pathway is activated resulting in a different shape of the kinetic profile (Figure 1).

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Figure 1. Possible mechanisms in a nucleation-elongation process: homogeneous nucleation and secondary nucleation. Circles represent monomers and red edges indicate the nucleus (5 units in this case). In particular, nucleation on already formed polymer surfaces is shown as an example of secondary pathway. On the right, temporal behavior for homogeneous and secondary nucleation as predicted by the mathematical approach of Ferrone and coworkers.

In all the above mentioned cases, the rate of polymer growth is largely enhanced compared to the simple homogeneous nucleation. To mathematically describe such increase, a positive term need to be added in the equation 1 and, reasonably, a linear dependence on the already polymerized monomer can be taken into account for all of the three possibilities. As a consequence, the following differential equation for secondary nucleation can be used:

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d cp dt

= J * ⋅c * +Q[c0 − c (t )]

(5)

where Q[c0-c(t)] is the term that takes into account the auto-catalytic processes. Using again the pertubative approach and coupling equation 5 with 2, the analytical solution is:

[

]

[c0 − c(t )] = A' cosh(B ' t ) − 1

(6)

where in this case A' and B' are functions of the rate constant, nucleus concentration and Q term. In the early stages, function in equation 6 resembles a homogeneous nucleation and afterwards a prompt increase in the growth rate occurs. As shown in the right side of Figure 1, an exponential function can also describe this temporal behavior with a good approximation. Noticeably, such a kind of behavior is entirely determined by the term Q[c0-c(t)] in equation 5.

1.2. Model System, Experimental Approach and Aim of the Study Insulin is a protein hormone largely used as a model system for the study of amyloid formation [29-31]. In fact, under specific conditions, i.e. high temperature and low pH, it is Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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very prone to form amyloid fibrils. Moreover, the fibrillogenesis pathway for insulin results in a highly diversified scenario both for the involved mechanisms and the occurring intermediate and final species [32-38]. Generally, insulin fibrillation kinetics are characterized by a long lag phase and a fast fibril growth and it is now assessed that secondary nucleation mechanisms determine such temporal profile [32, 33, 37] In particular, a crucial role is likely played by the surfaces of already formed fibrils able to act as nucleation sites. Such suggestion was reported first by Waugh [39, 40] and recently confirmed by means of imaging tools [32] so that insulin fibrillation can be considered a highly autocatalytic process. Moreover, it is worth noting that the kinetics profile can be affected by different experimental conditions, diverging from the classical three-steps curves (lag phase, rapid growth and saturation) and showing a range of different species in solution [38]. Several studies have shown that different amyloid morphologies may occur during aggregation [32, 34-36] as a consequence of different fibrillation pathways, strictly dependent on solvent composition as well as on hydrostatic pressure [38]. In acetic acid solutions the nucleus structure for insulin has been recently elucidated by means of Small Angle X-ray Scattering (SAXS), and the authors proposed a novel mechanism for fiber elongation, involving a structural nucleus which is also the elongating unit of fibrils [41]. Further, great interest has been also addressed to the effects of the experimental conditions on in vitro insulin fibrillation and to the intrinsic uncertainty of the kinetic parameters, underlying a high degree of stochasticity for this process [34, 37]. Thioflavin T (ThT) is a fluorescent dye widely used to “in situ” monitor amyloid fibril presence and growth, and we used it as a main tool to follow insulin fibrillation in the presented studies. ThT was introduced for studying amyloid formation in 1959 [42] and its spectroscopic properties in staining different typologies of materials and tissues, such as cartilage matrix, elastic fibers and mucopolysaccharides [42, 43] cellulose matrix, [44] DNA [45-47] and single-stranded polynucleotides [48] have been investigated in detail. ThT has a high selectivity for amyloid fibrils and when bound to amyloid aggregates, using an excitation wavelength of ~ 440-450 nm, shows a bright fluorescence emission in the 475-600 nm region. In contrast, in aqueous solution and in the presence of native proteins with different primary and secondary structure, ThT is characterized by a low emission quantum yield [49, 50]. Moreover, ThT does not affect [29, 51] the early formation of fibrils, making this method suitable for in situ fibril detection. The binding mechanisms of this dye with amyloidal structures, certainly depending on its molecular structure, have not been completely elucidated. ThT molecule is formed by three different main groups: the benzothiazole group, the benzene ring and the dimethylamino group (Figure 2). The spatial orientation of these three groups with respect to each other mainly determines both the specific interaction with aggregates and the spectroscopic properties of ThT [52-54]. The increased interests on this molecule is revealed by several recent studies on the mechanism of ThT-fibril interaction [55-58] and β-sheet structured cavities and channels, which are common structural features in amyloid fibrils, have been proposed as binding sites of ThT [55-57]. The binding was proposed to be highly directional, with the ThT long axis parallel to the elongation axis of the fibrils [57]. Moreover, it was found that the cavity size plays a crucial role in determining the characteristics of ThT fluorescence [55] and a reliable quantitative detection of fibril mass by ThT fluorescence should also take into account the accessibility of fibril surface and cavities [57]. Because of its chemical and optical stability

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under the chosen experimental conditions (high temperature and low pH) [59], in situ Thioflavin T staining was used for detection of the fibrillation process.

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Figure 2. Geometry optimized structures of Thioflavin T. Roman numerals are related to hydrogen atoms belonging to different chemical groups in the molecules: dimethylamino group (I), two methyl groups (II and III) and the two aromatic rings (IV and V). Atom color: hydrogen (gray), carbon (green), nitrogen (blue), sulphur (yellow) and oxygen (red).

The aim of the present study is to independently elucidate different aspects of the insulin aggregation reaction using specific conditions. To do this, the thermally induced aggregation process of both human and bovine insulin was studied at low pH and high temperature. Using these two different sequences of insulin and two different solvent compositions allowed focusing on different aspects of the fibrillation process. Firstly, human insulin fibrillation has been thermally induced in acetic acid solutions. In these conditions the study of the kinetic parameters and the reproducibility of the process is greatly simplified allowing a detailed analysis of the molecular mechanisms involved. On the contrary, bovine insulin fibrillation in HCl solution has been reported as a quite complex scenario both for the occurring species and the phenomenology of the process with kinetic profiles highly affected by physico-chemical parameters. For the above reasons, using two different insulin sequences in two different experimental conditions is only intended to give an overview of the potential diversified processes taking place in insulin fibrillation slightly varying the experimental conditions.

2. Results and Discussion 2.1. Human Insulin in Acetic Acid Solutions A study on the concentration-dependence of human insulin fibrillation in 20% acetic acid (pH 1.8) was performed to analyze the molecular mechanisms involved in the reaction and is presented in this section. Under these environmental conditions insulin is monomeric [29, 30, 60]. Furthermore, it was recently shown by small angle X-ray scattering measurements that in such conditions, at a concentration of 5 mg/ml, the protein remains monomeric for all the

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duration of the lag-phase; afterwards, helical oligomers were observed which may constitute both a structural nucleus and the elongation species in the growth of fibrils [41]. Fibril formation was monitored by Thioflavin T (ThT) fluorescence. In order to examine for unsought effects on fibrillation kinetics due to the presence of ThT we compared experiments in the absence of the dye with experiments performed at different ThT concentrations: the fibrillation process was assessed by the observation of the time evolution of turbidity of the samples. Moreover, to further characterize the reproducibility of the process by ThT fluorescence, we also performed a statistical study at three different insulin concentrations. Details about the experimental methods and sample preparation are reported in reference [61]. 2.1.1. Characterization of Fibril Formation

ThT Fluorescence (Arb.Un.)

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The time evolution of ThT fluorescence for samples of different protein concentrations in the range 1-20 mg/ml, at 45°C is reported in Figure 3. ThT concentration was kept constant in each sample and was 20 μM. Data show a characteristic sigmoidal profile: all the kinetics present a pronounced lag time, in which apparently nothing happens, followed by an abrupt increase of the fluorescence signal. It is noticeable that at each concentration in the selected experimental conditions, there is no evidence of the biphasic fibrillation behavior as observed by Grudzielanek et al. at analogous pH values for bovine insulin, when the protein is dissolved in aqueous solution (without acetic acid) [38].

10000

20 μM ThT 20mg/ml 15mg/ml 10mg/ml 7.5mg/ml 5mg/ml 2.5mg/ml 1mg/ml

8000 6000 4000 2000 0 0

2

4

6

8

10

12

14

16

18

20

Time (hours) Figure 3. Kinetics of human insulin fibrillation in 20% acetic acid, 0.5 M NaCl, 45°C (λexc=450nm λem=480nm). The ThT fluorescence is shown as a function of incubation time at different insulin concentrations.

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Furthermore, in line with other studies [29, 30, 33], Figure 3 shows a decrease of the observed lag time by increasing insulin concentration, at least in the range 1-5 mg/ml. It was verified that increasing concentration of ThT in the range 5 and 40μM did not alter the fibrillation kinetics. To describe the main features of the observed fibrillation process two characteristic parameters were selected: 1) ThT fluorescence plateau value (FFV, see Figure 4A), and 2) the reciprocal of the time necessary to reach 50% of FFV ( 1 t50% , see Figure 4B). These values are shown in Figure 4A and B as a function of protein concentration, for three different concentrations of ThT (5, 20 and 40 μM). As can be seen for both parameters a sort of saturation effect for protein concentrations exists above ~ 5 mg/ml. Concerning FFV (Figure 4A), the saturation level clearly depends on the amount of ThT in the sample. Saturation effect by itself cannot be ascribed to experimental artifacts such as insufficient concentration of ThT in solution with respect to the aggregate number. To verify this statement, we performed a series of measurements at high protein concentration (20 mg/ml) and ThT concentrations spanning from 2.5 to 80 μM. The dependence of FFV on ThT concentration for these measurements is shown in Figure 5; the results (open triangles) display a linear dependence of FFV on concentration from 2.5 μM up to 10 μM. For larger concentrations, starting from 20 μM of ThT, the FFV value is almost unaffected by further addition of the dye. It is worth to note that, the effects of the absorption of the fluorescence excitation beam at different ThT concentrations in the bottom-bottom configuration were estimated and corrected [61]. The corrected data are also shown in Figure 5 (full circles) confirming that the observed saturation effect at higher ThT concentration is mainly ascribable to the peculiarity of fibril-dye binding. It was also verified that in these experimental conditions insulin fibrillation proceeds by an essentially total conversion of native protein into amyloid aggregates [41]. In view of these observations, the behavior of data in Figure 4A and B, at least for 20 μM and 40μM ThT, seems to be representative of an intrinsic property of the fibrillation process. It is possible to infer that above a protein concentration of 5 mg/ml, the number of binding sites in the fibrils for ThT yielding the characteristic fluorescence remains almost constant. The values of 1 t50% (Figure 4B) are barely affected by the amount of ThT in solution. Similar values were also obtained in the absence of ThT, by measuring the progress of the fibrillation process by sample turbidity (data not shown). It is noticeable that on average, increasing the ThT concentration results in a slightly slower fibrillation process, without changing the main features of its protein concentration dependence and of the above mentioned saturation effect. Importantly, the four replicates for each measurement indicate that the reproducibility of the fibrillation process significantly improves at high protein concentration (see the error bars in Figure 4A and 4B). Again, it will be shown that this behavior is representative of an intrinsic property of fibrillation.

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ThT FFV (Arb. Un.)

10000

(A)

8000 6000 4000 2000

1/t50%(hours -1 )

0 0.35

(B)

0.30 0.25 0.20 0.15

40 μM ThT 20 μM ThT 5 μM ThT

0.10 0.05 0.00 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

Human insulin concentration (mg/ml) Figure 4. (A) ThT fluorescence final value (FFV) as a function of protein concentration at three different ThT

t50% ) as

a function of protein concentration at three different ThT concentrations. Error bars represent absolute deviations observed on four replicates.

20 mg/ml Human Insulin

12000

ThT FFV (Arb.Un.)

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concentrations. (B) Inverse of the time at which the fluorescence signal reaches 50% of the FFV ( 1

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In Figure 6, the same kinetics shown in Figure 3 are reported after scaling fluorescence intensity and incubation time for FFV and t50% values, respectively. All the kinetics in the

Normalized ThT Fluorescence Value

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range 2.5 – 20 mg/ml scale well, especially for the first half of the trace. As evident data obtained at 1 mg/ml have a different trend with respect to the others and also show a lower reproducibility, indicating the presence at this concentration of some unpredictable effects, probably enhanced by the long duration of the experiment (~ 20 hours). It is important to note that the scaling properties highlighted in Figure 6 are not trivial. Indeed, the duration of the lag phase, and the rate constant of fibril growth here chosen as suitable parameters to describe the fibril formation kinetics are not necessarily related to each other. The scaling of data suggests there actually is a correlation between these two parameters; as recently reported, such a correlation may be a common property in the formation of amyloid aggregates [62].

20mg/ml 15mg/ml 10mg/ml 7.5mg/ml 5mg/ml 2.5mg/ml 1 mg/ml

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t50%

in the

abscissa.

It must be noted that the lag time values and rate constants of fibril growth are highly dependent on the experimental setting (e.g. plate or cuvette material, surface-volume ratio of the sample, stirring and small unpredictable impurities in solution) and then may not be “absolute.” Regarding the effect of experimental methods on nucleation events, several insights have been recently provided for insulin amyloid formation [34, 37], in particular, it has been observed by differential interference contrast (DIC) optical microscopy that glass surface defects or not properly cleaned glass surfaces catalyze the formation of amyloidogenic spheroid structures with the result of affecting dramatically both the subsequent nucleation and elongation mechanisms [34]. Furthermore, it was recently shown for islet amyloid peptide that the first nucleation events seem to be fully controlled by interactions between the protein molecules and liquid-liquid or liquid-solid interfaces, which act as nucleation points via a surface-assisted process [23]. Thus, different experimental

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settings may induce different protein-surface interactions, thus resulting in a significant variability in the process parameters as determined by different investigators. 2.1.2. Correlation between FFV and 1/t50% The scaling properties displayed in Figure 6 indicate that one kinetic parameter suffices to describe the fibrillation process, and that the value of 1 t50% (Figure 4B) can be used as a

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sort of overall rate constant to take in account for the velocity of the process. In Figure 7, this parameter is reported as a function of the FFV. Quite interestingly, as shown in the figure for 20 μM and 40 μM ThT concentrations, a linear relation exists between these two parameters, while none of them show a linear dependence on protein concentration (see Figs. 4A and B). The observed correlation, again, is not obvious at all, and the fact that it is observed at different ThT concentrations strongly suggests that it is not accidental. On the contrary, it indicates the existence of some meaningful physical quantity in the process, which determines both parameters, and therefore the overall profile of the kinetics. In agreement with the existence of secondary nucleation for insulin [32, 33, 63, 64] and with the recently proposed mechanisms for ThT binding to fibrils [55-57], this quantity can be identified as the accessible surface of fibrils: a larger accessible surface brings about a larger number of nucleation sites, promoting an enhanced growth of new fibrils. Moreover, at the same time, larger accessible surfaces mean a larger accessibility for ThT to binding sites. Accordingly, both the overall rate constant of the process ( 1 t50% ) and the ThT fluorescence intensity (FFV) should be proportional to the accessible surface of fibrils and therefore linearly related to each other (Figure 7). Concerning the concentration dependencies of the parameters shown in Figure 4A and B, it must be noted that, in general, different aggregate morphologies may occur in protein amyloid aggregation [34, 37, 38]. For example, for insulin fibrillation, different environmental factors, i.e. solvent properties [30, 35, 37], cosolvents addition [36, 38, 65] and pressure [38] may affect the overall aggregation pathway [58] and the spatial arrangement of mature fibrils [34, 37, 65]. Different fiber morphologies may result in a different ratio of fiber accessible surface and mass; this makes critical to determine a reliable estimation of the amyloid mass by ThT essay [37]. Several different structures of aggregates have been observed during the various phases of insulin fibrillation [34, 37]. As clearly pointed out by Manno et al. [37], fibrils of different thickness can be formed and assembled in bundles, with a reduced accessible surface with respect to thin filaments. Our data indicates that above ~5 mg/ml fibril formation may be affected by a sort of crowding effect which is due to increased collision probability and aggregates growth; this may favor the formation of less accessible regions to the dye. Such a scenario may explain the concentration saturation effect shown in Figure 4A and B for both FFV and 1 t50% . A role could also be played by the observed tendency of morphologically distinct templates to replicate themselves in an autocatalytic way [66, 67].

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0.35

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ThT FFV (Arb.Un.) Figure 7.

1 t50%

as a function of FFV for all the investigated protein concentrations at two different ThT

concentrations. Error bars represent absolute deviations observed on four replicates.

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It is worth to note that the saturation effect shown in Figure 4B for 1 t50% , i.e. the weakening of the concentration dependence of the rate observed for fibril formation and growth, may have other alternative explanations. In particular, Powers and Powers (2006) recently showed that it arises from the fact that above a “supercritical concentration” oligomers population may become significant with respect to the total protein concentration [68]. When this happens, if aggregates can only grow by monomer addition, the rate of fibril growth may become almost independent of protein concentration because of monomer conversion to oligomers. This explanation however does not seem applicable in the observed process. In fact, in the case of insulin, fibrils may grow not only by monomer addition, but also by oligomer addition indeed. Although small angle X-ray scattering measurements have shown the existence of a large population of oligomers, they were found to constitute both a structural nucleus and an elongation species in the growth of fibrils [41]. As a result, oligomer formation does not explain that the lag time no longer decreases at high protein concentration. However, the rate of formation of these oligomers may also be affected by a secondary nucleation mechanism, i.e., by the accessible surface of the fibrils. The role of fibril surface was first noticed several decades ago by Waugh [39, 40], who proposed a model in which the rate of growth of fibrillated material was proportional to the fibril surface area [40]. However, in secondary nucleation processes what is proportional to the surface of fibrils is not the rate of growth of fibrillated material, but, instead, the rate of formation of new fibrils. The two models are conceptually different and they lead to distinct differential equations for the description of the kinetics [22, 40]. 2.1.3. Statistical Study In Figure 4 it was highlighted that the reproducibility of the fibrillation process sizably increased with protein concentration and this was suggested to arise from an intrinsic property of insulin fibrillation in the observed conditions. In order to deeply elucidate this issue, we Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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carried out series of experiments with the aim to make a statistical investigation on samples at three different insulin concentrations (1, 2.5 and 5 mg/ml, see reference [61] for the experimental setting). As evident in Figure 8, apparently identical insulin samples, when incubated at 45°C, do not show exactly the same kinetic profile. In Figure 9 we present the statistical distributions of both FFV and 1 t50% normalized for their average value, this operation makes clear the properties of the different dataset: the relative spread of the data clearly depends on insulin concentration, being larger at lower protein concentration. In general, the formation of the early aggregates in solution may be considered an inherently stochastic event [22], resulting in some level of variability of the process parameters. In particular, the spread of 1 t50% in our data is mainly determined by a variation in the lag time

7000

ThT Fluorescence (Arb.Un.)

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values and not in the elongation rate (analysis not shown), suggesting a process initially controlled by stochastic events. As recently observed for the aggregation kinetics of βamyloid peptide [69], the kinetic reaction appears to be more reproducible at high protein concentration and, in general, in conditions favoring intermolecular association. A large variability in the lag phase of the HbS polymerization process has been observed by Hofrichter (1986), who proposed that the early stages of the kinetics are fully determined by a single nucleation event and by a subsequent fast growth of polymers, explained, as for insulin, by secondary nucleation mechanisms [70]. In this sense, insulin fibrillation seems to resemble HbS polymerization: a single nucleation event may happen in a given sample region and then the first stable aggregates determine the further growth of fibrils, in accordance with the spatial heterogeneity recently proposed for insulin fibril formation [63]. However, it is important to stress again that the nature of the lag-phase strongly depends on the environmental conditions. In fact, in other solvents, ThT fluorescence shows that the lagphase may be entirely suppressed [37], and even a biphasic fibrillation pathway may occur [38] , with an essentially immediate “pre-transition.”

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Nucleation Mechanisms and Morphologies in Insulin … 25 (A)

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Figure 9. Statistical distribution of FFV and

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50%

/

1 t50% normalized for their average value. (A) 5 mg/ml, (B) 2.5

mg/ml and (C) 1 mg/ml of human insulin.

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2.2. Bovine insulin in HCl Solutions Thermally induced fibril formation in bovine insulin samples has been also studied in HCl solution. With the aim of studying the effect of protein concentration on aggregation mechanisms, progress curves for fibril formation at different insulin concentrations were detected using the fluorescent amyloid-selective dye Thioflavin T (ThT). Further, atomic force microscopy has been used to identify different morphologies along the fibrillation pathway. Finally, dynamic light scattering together with ThT assay have been also used for the study of the early stages of the process in the low protein concentration regime. For this study, evolution of the whole ThT spectrum has been revealed using a CCD camera with a high sensitivity and a fast acquisition time leading to the record of 1 spectrum per second. This temporal peculiarity allowed a quantitative detection of the signals in the early stages of the process, pointing out a pronounced spatial heterogeneity in the formation of the early stable aggregates. Details about the experimental methods and sample preparation are reported in reference [71]. 2.2.1 Different Processes and Morphologies Occurring during the Fibrillation Kinetics A further analysis of the effect of insulin concentration was performed on bovine insulin sample using solutions containing HCl. Figure 10 shows the progress curves of ThT fluorescence as a function of the incubation time at different insulin concentrations, ranging from 0.5 to 10 mg/ml, incubated at 60°C with 20 μM of ThT. At low concentrations (< 3 mg/ml), the kinetics present the well known sigmoidal three steps behavior with a pronounced

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lag phase, a subsequent very fast growth of amyloid aggregates, and a final saturation, as revealed by ThT intensity. Interestingly, in solutions at analogous low pH and in presence of acetic acid, both human insulin (see Figure 3) and bovine insulin [33] present a classical three-steps curve, being such a profile a common feature in a wide range of protein concentrations (1-20 mg/ml). On the contrary, as shown in Figure 10, in presence of HCl the increase of insulin concentration produces drastic changes in the kinetics profile. ThT emission shows a biphasic temporal behavior in the intermediate range of concentration investigated (3.5 and 4 mg/ml) and an almost absent lag phase at higher concentrations (5-10 mg/ml). It is worth of note that, when bovine insulin is dissolved in aqueous solution at low pH (without acetic acid), the appearance of a double process has been already reported by Grudzielanek and coworkers [38]. In that study, the occurrence of two concurrent aggregation pathways had been triggered by tuning the pressure and adding salt. The authors proposed a scheme in which formation of an intermediate species may determine the first increase of ThT intensity (pre-transition) and afterwards the cooperative mechanism leads to the formation of mature fibrils (main transition). Further, during the formation of the intermediate species (i.e. oligomers), they also revealed a conversion of the native secondary structure to a β-sheet-rich structure [38]. Based on the proposed binding model of ThT to β−sheet cavities [55], presence of such secondary structure in the oligomers is also in agreement with the enhanced ThT fluorescence obtained [38]. Noticeably, our data show that, in HCl solutions, the occurrence of the so-called “pre-transition” and “main transition” phases may also be dependent on insulin concentration. In particular, they simultaneously occur only in the range of insulin concentration of 3-4 mg/ml. With the aim of further studying this double process, an investigation on the different species occurring during the fibrillation pathway at 4 mg/ml as a representative concentration has been carried out. The morphologies of the aggregates at different incubation times were investigated by atomic force microscopy as indicated by the arrows and roman numerals in Figure 11a. Scans are displayed in Figure 11b. As expected, the sample at 25°C before incubation (Figure 11b, I ) does not show sizable objects in solution and only species with a height of 1-2 nm are revealed, as also confirmed by dynamic light scattering analysis (data not shown). In agreement with previous AFM observations [32] and small angle X-ray scattering data [29], this indicates a major presence of insulin monomers and dimers in low pH solutions with HCl. After 40 minutes of incubation (Figure 11b, II) and in correspondence of the first phase of the ThT intensity growth (see Figure 11a), small protein assemblies are present in solution. Interestingly, a clear presence of such population has been also revealed during the kinetics by dynamic light scattering measurements and a hydrodynamic radius of ~ 15 nm has been estimated for this species (not shown). Such assembled molecules are not elongated and resemble the oligomers observed by Grudzielanek and coworkers [38] Moreover, the ThT-positive staining of this intermediate (Figure 11a) strongly suggests the presence of cavities rich in β-sheet structures with a proper size able to hold the entire ThT molecule inducing the characteristic fluorescence [55]. After ~60 min, together with oligomers, thin filaments (2-5 nm in height) start appearing in the solution. Such fibers display an unstructured shape and a length of 50-300 nm. In this sample, evidences of branching are not observed (Figure 11b, III). After 90 minutes of incubation, at the first ThT fluorescence plateau (Figure 11b, IV), fibers of 0.5-3 microns in length with an almost unvaried height are observed. In the following, at the beginning of the second ThT increase phase, after 140 minutes (Figure 11b, V), few branched fibers are observed in the

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Normalized ThT

sample evidencing the activation of new nucleation/elongation events in proximity of already formed fibrils: these events can be classified as secondary nucleation, (see zoom of Figure 11b, V). A noticeable increase in the thickness up to 9 nm is also observed in this sample. Such trend is more pronounced in the subsequent part of the reaction up to the formation of a highly branched and widely distributed network of fibrils at the end of the kinetics (~ 180 min, Figure 11b, VI and related zoom). Moreover, besides the branching, in the last part of the kinetics it is possible to observe thicker fibrils with detected height up to ~ 12 nm possibly grown via the interaction of fibrils surfaces or superimposition of already formed fibrils (see zoom of Figure 11b, VI), confirming the diversity of fiber morphologies along the fibrillation pathway [32, 37]. At high concentrations (> 4 mg/ml), besides the absence of the lag time, the biphasic behavior does not take place or, probably, the occurrences of the two processes could occur too fast to be clearly detected. 1.0 0.5 mg/ml 1 mg/ml 2 mg/ml 2.5 mg/ml 3 mg/ml 3.5 mg/ml 4 mg/ml 5 mg/ml 6 mg/ml 10 mg/ml

0.8 0.6 0.4 0.2 0.0 0

1

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Time (hours) Figure 10. Kinetics of bovine insulin fibrillation in 25mM HCl, 0.1 M NaCl, 60°C (λexc=450 nm λem=480 nm). ThT emission normalized to the plateau value is shown as a function of incubation time at different insulin concentrations.

2.2.2. Lag Phase in the Low Concentration Regime For concentrations < 3 mg/ml, a standard three-steps mechanism seems to take place with a decreasing lag phase when protein concentration is increased, and without any evidence of biphasic behavior (Figure 10). We mainly focused on the early stages of the process starting with the analysis of the lag phase during fibrillation at low protein concentrations (< 3 mg/ml). Lag phase is generally indicated as the time in which apparently nothing happens and no significant changes in the detected signals are revealed, as evidenced, for example, in our measurements at 0.5 mg/ml in Figure 10 (~ 4.5 hours). However, we showed that significant events are detectable within the lag phase and that they have a relevant role in the aggregation pathway. To further investigate the lag phase, we detected the ThT emission spectra using a high sensitive CCD camera [71] and dynamic light scattering signals.

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Normalized ThT

1.2 1.0

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(b)

Fiigure 11. Fibrilllation kinetics of o 4 mg/ml bovvine insulin in 25 mM HCl, 0.1 0 M NaCl, 600°C. (a) ThT fluuorescence curv ve during the proocess (b) SFM im mages at differeent instants of thhe kinetics. Rom man numerals inndicate samples at different incuubation times ass shown in Figuure 11a. (I) 0 miin, (II) 40 min, (III) 60 min, (IV V) 90 min, (V) 140 min, (VI) 180 1 min. In the blue dotted boxxes, zooms on thhe fibril morphoologies in the finnal part of the kiinetics are reportted.

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Scattering (kcps)

ThT (Arb.Un.)

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Figure 12. (a) ThT fluorescence and (b) DLS intensity as a function of the incubation time during the early stages of fibrillation of 0.5 mg/ml bovine insulin in 25 mM HCl, 0.1 M NaCl, 60°C. Solid lines are quadratic fitting curves of data during the lag phase (primary nucleation). Dashed lines are exponential growth fitting curves of the whole data set (secondary nucleation).

As shown in Figure 12a for the kinetics at 0.5 mg/ml of bovine insulin, the ThT signal is not exactly constant during the “nominal” lag. phase and shows a very slight increase since the first instants up to ~ 4.5 hours of incubation. Such intensity increase, even if very low, is highly reproducible on several independent attempts. Afterwards, i.e. when secondary nucleation mechanisms take the control of the reaction, ThT emission is enhanced of several orders (up to ~ 100 times of the initial value). Moreover, the kinetic profile of the dynamic light scattering signal shows the same behavior (Figure 12b). We note that the above reported low increase in the early stages has been obtained for samples up to 2.5 mg/ml of insulin concentration. Such data could suggest that primary (homogeneous) nucleation takes place during the lag phase, whose temporal length is related to the onset of secondary nucleation. In fact, at the beginning of the fibril formation, elongation of small fibrils can be considered as a sequence of a monomer addition of unfolded or native species to the nuclei. Such a reaction can be rationalized in terms of the homogeneous nucleation, leading to a quadratic time dependence [22] (solid lines in Figures 12a and 12b and Figure 1) and to lower ThT quantum yield. Afterwards, when fibrils size increases, exposed fibril surfaces allow the onset of the secondary nucleation [32, 33] giving rise to an exponential growth of fibrils [22] (dashed lines in Figures 12a and 12b). However, it must be noted that the initial signal increase in Figures 12a and 12b could also be ascribed to a residual formation of β-sheet oligomers, as those observed at higher concentration (Figures 11a and 11b). In fact, a very low concentration of

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such species could also contribute to the initial slight increase of the signals in the kinetics at insulin concentration < 2.5 mg/ml, and their formation, even if not clearly detected by DLS analysis (data not shown), cannot be excluded. It is also worth to note that in this protein concentration regime, an increase of the ThT signal since the early stages of the process has also been observed by Manno and coworkers [37] and a detailed analysis of the early stages by means of high resolution atomic force microscopy has also shown a general tendency of the insulin molecules to cluster and form small pre-fibrillar species in the first instant of the kinetics, the latter probably contributing to the ThT/scattering slight increase (Figures 12a and 12b) [32]. All these evidences confirm that, at least in HCl solutions, the period before the onset of sizable fibrils is far from being “flat” [37] and need to be further investigated.

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2.2.3. Initial Fibrils Growth in the Low Concentration Regime In our previous work [63], we pointed out that during fibrillation of bovine insulin at protein concentrations < 1 mg/ml and in HCl solution, ThT emission spectra at the end of the lag phase present a highly distorted signal as revealed by a PMT detector (for an example see Figure 2 in Librizzi et al. 2007 [63]). Moreover, such fluctuations result in being mostly suppressed for insulin fibrillation in confined environment where protein mobility is strongly reduced [63]. With the aim of further characterizing such peculiarity and clarifying the involved mechanisms, we detected the early stages of ThT intensity growth using a CCD camera [71]. By means of this acquisition system we were able to quantitatively detect the intensity fluctuations of the emission spectra as a function of the protein concentration (< 2.5 mg/ml). By a highly focalized beam (λexc=450 nm), a portion of the sample volume has been excited and emission spectra have been detected (440-650 nm). As an example, Figure 13 shows the time evolution of the emission spectra in the last part of the lag phase (0.5 mg/ml of bovine insulin after ~5 hours of incubation) detected with an acquisition time of 1 sec per spectrum for a 5 minutes. Spectra present a narrow band at 450 nm (elastic scattering, being its intensity related to the molecule sizes in solution [72]) and a lower intensity and broader band centered at 480 nm due to the ThT fluorescence, i.e. occurrence of fibrils or, generally, channels rich in β−sheets structures [55]. In the temporal window considered, both the signals start to increase and show large intensity fluctuations in correspondence of the two peaks, being such fluctuations evidenced by the red lines in Figure 13. Such intensity jumps are quite reproducible both in amplitude and in temporal length for different fibrillation kinetics. The single-wavelength kinetics profile both for ThT (Figure 14a) and elastic scattering (data not shown) as a function of the incubation time have been considered with the aim of quantitatively studying such details of the kinetics. At the end of the lag phase and during the initial growth phase, intensity starts to sizably increase and displays local large deviations from a standard three-steps curve (see best fit function in Figure 14a and reference [71] for details). In Figure 14b we report the time evolution of these deviations with respect to the best fit curve. During the lag phase no significant oscillations of the signal have been detected, whereas fluctuations up to the ~15% of the fluorescence final value are evident during the initial growth of fibrils (Figure 14b). Afterwards, ThT intensity linearly increases reaching a plateau at the end of the reaction without any further jump in the emission signal.

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Nucleation Mechanisms and Morphologies in Insulin …

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Figure 13. 300 emission spectra (λexc= 450nm) as detected for 300 seconds at the end of the lag phase (16800-17100 seconds of incubation) of 0.5 mg/ml of bovine insulin in 25 mM HCl, 0.1 M NaCl at 60°C (20 μM ThT). Red lines are indicated to better visualize the evolution of elastic scattering (λem= 450nm) and ThT intensity (λem=480nm).

Our experimental data suggest that at the end of the lag phase large aggregates are already present in some part of the sample volume (spatial heterogeneity). Formation of stable aggregates in a given region of the sample, i.e. single nucleation events, can be considered intrinsically stochastic [22], being spatially localized in the sample and temporally independent from each other [70]. The small aggregates diffuse through the solution and, crossing the incoming beam produce fluctuations both in the elastic scattering and ThT signal (Figure 13 and Figure 14a and 14b). As shown in Figure 14c, elastic scattering and ThT signals show a linear correlation for the entire length of the incubation. Consequently, all the sizable objects crossing the volume and producing fluctuations in the scattering signal seem to have an amyloidal origin or, at least, a high β-sheet content. Further, supporting our hypothesis of the “diffusing aggregates,” intensity fluctuations are mostly suppressed by increasing the illuminated sample volume, i.e. using small sample volume (~100 μl) or taking out the focalizing lens system (see reference [71] for details). It is also worth to note that intensity fluctuations have not been revealed during aggregation in the same conditions of other proteins where a spatially homogeneous nucleation occurs (e.g. Concanavalin A) [19]. The same kind of measurements and analysis as those shown in Figure 14a has been carried out for 4 different insulin concentrations up to 2.5 mg/ml. Increasing the protein concentration, both the temporal length and the amplitude of the fluctuations decrease. To quantitatively estimate the fluctuations we considered the total amplitude of square residuals for each concentration in the initial growth phase (see reference [71] for details on the analysis). Figure 14d clearly shows that above 1 mg/ml no significant fluctuation occurs, whereas for lower concentrations (< 1 mg/ml) diffusion of early stable aggregate seems to be one of the main events at the beginning of the growth phase.

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(a)

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3.0

Insulin Concentration (mg/mL)

Figure 14. (a) Normalized ThT intensity during kinetics of 0.5 mg/ml bovine insulin in 25 mM HCl, 0.1 M NaCl, 60°C (λexc=450nm λem=480nm). Solid line represents the best fit function (see reference [71]). (b) Temporal profile of percentual residuals as obtained by fitting of the experimental data in Figure 14a. (c) Normalized ThT intensity (λexc=450nm λem=480nm) as a function of the normalized elastic scattering intensity (λexc=450nm λem=450nm) during the kinetics of fibril formation of 0.5 mg/ml bovine insulin in 25mM HCl, 0.1 M NaCl, 60°C. Solid line represents a linear fitting of 1300 experimental data points. ThT and elastic scattering were simultaneously measured (see text). (d) Thioflavin T fluctuations as a function of the insulin concentration. Solid line is a guide for eyes.

Data in Figure 14d can be explained in the framework of the secondary nucleation mechanisms proposed for insulin fibrillation [32, 33]. In presence of such a process, first stable fibrils can act as nucleation sites for the protein molecules in solution, i.e. by accessible surface of fibrils (see previous sections). As a consequence, after the first nucleation events and during the diffusion of early aggregates, secondary pathways take the control of the kinetics, determining the overall growth of amyloid aggregates in the whole sample volume. The increase of protein concentration in the sample leads to a higher probability of primary nucleation events and of interactions between protein molecules and already formed fibrils. This produces a faster establishment of a spatially homogeneous distribution of amyloid fibrils in the whole sample volume. Such a mechanism probably determines the damping, both in the amplitude and in the temporal length, of the intensity fluctuations at higher protein concentration (Figure 14d). Furthermore, in agreement with the statistical investigation reported above for human insulin fibrillation in acetic acid (Figure 9) and with the aggregation kinetics of β-amyloid peptide [69], we registered a higher reproducibility of the fibrillation kinetics profiles, i.e. lag time and growth rate, at higher protein concentration also for insulin in HCl solutions (data not shown). This evidence can be rationalized in terms of the same mechanism that leads to the damping of fluctuations, so that spatial heterogeneity in

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the formation of early stable aggregates, i.e. single stochastic nucleation events, represents the main factor determining the degree of reproducibility of the kinetic parameters. All the above considerations supported by data in Figure 14d strongly suggest a connection between the molecular mechanisms, i.e. secondary nucleation mechanism and diffusion of aggregates in solution, and the experimental reproducibility of the fibrillation kinetics profiles.

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Conclusions In this chapter, we presented an experimental study on insulin amyloid fibril formation. Inducing insulin fibril formation slightly changing the solvent properties and the temperature revealed different aspects related to the phenomenology of the fibrillation reaction. Firstly, using ThT fluorescence final value (FFV) and the inverse of the time at which the fluorescence signal reaches half of the final value (1/t50%), we described the kinetics in a wide range of concentrations. The linear relationship between FFV and 1/t50% indicates that the fibrillation process is essentially determined by a fibril surface-catalyzed mechanism, through secondary nucleation pathways. Moreover, our data show that, at least in early stages, the process is inherently stochastic and is characterized by an intrinsic variability of the kinetic parameters. Secondly, we also performed an experimental study on insulin amyloid fibril formation in HCl solution. Our data clearly show that, in these conditions, the presence of two different self-assembling pathways can be triggered by varying protein concentration. In fact, a doublesigmoidal process occurs at 3.5-4 mg/ml. The biphasic profile reveals the presence in the early stages of an intermediate species, indicated as oligomers, able to bind to ThT inducing the characteristic fluorescence. Interestingly, such oligomers are clearly detected by AFM and dynamic light scattering only in a narrow range of protein concentration and a characteristic hydrodynamic radius of ~15 nm has been estimated by the analysis of the dynamic light scattering data. In the late stages of the process, different morphologies of elongated fibrillar species, i.e. fibers of different thickness and network of fibrils, are also revealed by AFM images. Moreover, an accurate analysis of the different stages in the low concentration regime (< 3 mg/ml) has been performed and ThT and dynamic light scattering signals show a slight increase in the intensity since the first instants of incubation. Then, ThT fluorescence and elastic scattering display large intensity fluctuations dependent on insulin concentration and afterwards the signals increase of several orders of magnitude. Such a sequence of events may suggest a complex scenario with several interconnected steps as presented in the illustrative and simplified scheme of Figure 15. After a first step in which a partial unfolding of the native state (not clearly detected yet) probably occurs, primary (homogeneous) nucleation takes place in the early stages of the process, followed by a stochastic (temporally and spatially) formation of the first stable aggregates that, diffusing through the solution, produces the detected intensity fluctuations. Eventually, stable aggregates likely promote the formation of amyloid fibrils in the whole sample volume by secondary nucleation mechanisms, determining the exponential growth of the detected signals. We want to remark that the experimental approach here employed for the study of the spatial heterogeneity could be of general use for revealing stochastic nucleation events in amyloid protein aggregation or in polymerizing systems. Further, the revealed spatial heterogeneity points out a general

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problem on how crucial the choice of the experimental settings could be, i.e. the ratio between the illuminated volume and the total sample volume, for the reliability of the detected temporal features of such a kind of reactions.

Figure 15. Scheme for insulin fibrillation mechanism in HCl solution and in the low protein concentration regime as suggested by experimental data in Figures 12-14. In this simplified representation, no distinction was made among the possible different morphologies and sizes of fibril at the end of the process (thin filament, bundles, branched fibrils, etc.) and they are all represented as green intertwined cylinders

Acknowledgments We thank Sven Frokjaer, Marco van de Weert, Minna Groenning and Bente Vestergaard, University of Copenhagen for useful discussions and scientific collaboration. We thank Novo Nordisk A/S, Denmark, for providing human insulin. This work was partly supported by a national project (PRIN2005) of the Italian Ministry of University Research and by POR Sicilia - Mis. 3.15-C.

References [1] [2]

Harper, J.; Wong, S.; Lieber, C.; Lansbury, P., Chem. Biol. 1997, 4, 119-25. Manno, M.; Emanuele, A.; Martorana, V.; Bulone, D.; San Biagio, P. L.; PalmaVittorelli, M.B.; Palma, M. U., Physical Review E 1999, 59 (2), 2222

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[35] Dzwolak, W.; Ravindra, R.; Winter, R. Phys. Chem. Chem. Phys. 2004, 6, 1938-1943. [36] Dzwolak, W.; Jansen, R.; Smirnovas, V.; Loksztejn, A.; Porowski, S.; Winter, R. Phys. Chem. Chem. Phys. 2005, 7, 1349-1351. [37] Manno, M.; Craparo, E.F.; Podestà, A.; Bulone, D.; Carrotta, R.; Martorana, V.;, Tiana, G.; San Biagio, P.L. J. Mol. Biol. 2007, 366, 258–274. [38] Grudzielanek, S.; Smirnovas, V.;Winter, R. J. Mol. Biol. 2006, 356, 497–509 [39] Waugh D.F. J. Am. Chem. Soc. 1946, 68, 247-250. [40] Waugh D.F., Wilhelmson D.F., Commerford S.L. and Sackler M.L. J. Am. Chem. Soc. 1953 75, 2592-2600. [41] Vestergaard, B.; Groenning, M.; Roessle, M.; Kastrup, J.S.; van de Weert, M.; Flink, J.M.; Frokjaer, S.; Gajhede, M.; Svergun, D.I. PLoS Biol. 2007, 5, 1089-1097 e134. [42] Vassar P.S. and Culling C.F.A. Arch. Pathol. 1959 68, 487-498. [43] Kelenyi G. Acta Neuropathol. 1967 7, 336-348. [44] Raj Retna C. and Ramaraj R. Photochem Photobiol. 2001 74, 752-759. [45] Parker A. and Joyce T.A. Photochem. Photobiol. 1973 18, 467-474. [46] Shirra R. Chem. Phys. Lett. 1985 119, 463-466. [47] Margulis L., Rozen H. and Nir S. Clays Clay Miner. 1988 36, 270-276. [48] Cundall R.B., Davies A.K., Morris P.G. and Williams J. J. Photochemistry 1981 17, 369-376. [49] Naiki H., Higuchi K., Hosokawa M. and Takeda M. Anal. Biochem. 1989 177, 244-249. [50] Levine III H. Prot. Sci. 1993 2, 404-410. [51] Levine III, H. Arch. Biochem. Biophys. 1997, 342, 306–316. [52] Voropai, E.S., Samtsov, M.P.; Kaplevskii, K.N.; Maskevich, A.A.; Stepuro, V.I.; Povarova, O.I.; Kuznetsova, M.; Turoverov, K.K.; Fink, A.L.; Uversky, V.N. J. Appl. Phys. 2003, 70, 868-874. [53] Maskevich, A.A.; Stsiapura, V.I.; Kuzmitsky, V.A.; Kuznetsova, I.M.; Povarova, O.I.; Uversky, V.N.; Turoverov, K.K. J. Proteome Res. 2007, 6, 1392-1401. [54] Stsiapura, V.I.; Maskevich, A.A.; Kuzmitsky, V.A.; Turoverov, K.K.; Kuznetsova, I.M. J. Phys. Chem. A 2007, 111, 4829-35 [55] Groenning, M.; Olsen, L.; van de Weert, M.; Flink, J.M.; Frokjaer, S.; Jorgensen, F.S. J. Struct. Biol. 2007, 158, 358–369 [56] Groenning, M.; Norrman, M.; Flink, J.M.; van de Weert, M.; Bukrinsky, J.T.; Schluckebier, G.; Frokjaer, S. J. Struct. Biol. 2007, 159, 483-497. [57] Krebs, M.H.R.; Bromley, E.H.C.; Donald, A.M. J. Struct. Biol. 2005, 149, 30-37. [58] Khurana, R.; Coleman, C.; Ionescu-Zanetti, C.; Carter, S.A.; Krishna, V.; Grover, R.K.; Roy; R.; Singh, S. J. Struct. Biol. 2005, 151, 229-238. [59] Foderà, V.; Groenning, M.; Vetri, V.; Librizzi, F.; Spagnolo, S.; Cornett, C.; Olsen, L.; van de Weert, M., Leone, M. J. Phys. Chem B 2008, 112, 15174-15181. [60] Smith, J.F.; Knowles T.P.J.; Dobson, C.M.; MacPhee, C.E.; Welland, M.E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15806-15811 [61] Foderà, V.; Librizzi, F.; Groenning, M.; van de Weert, M.; Leone, M. J. Phys. Chem. B 2008, 112, 3853-3858. [62] Fandrich, M. J. Mol. Biol. 2007, 365, 1266-1270 [63] Librizzi, F.; Foderà, V.; Vetri, V.; Lo Presti, C.; Leone M. Eur. Biophys. J. 2007, 36, 711-715.

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[64] Manno, M.; Craparo, E.F.; Martorana, V.; Bulone, D.; San Biagio, P.L. Biophys. J. 2006, 90, 4585-4591. [65] Grudzielanek, S.; Jansen, R.; Winter, R. J. Mol. Biol. 2005, 351, 879-894. [66] Dzwolak, W.; Smirnovas, V.; Jansen, R.; Winter, R. Prot. Sci. 2004, 13, 1927-1932. [67] Dzwolak, W.; Grudzielanek, S.; Smirnovas, V.; Ravindra, R.; Nicolini, C.; Jansen, R.; Loksztejn, A.; Porowski, S.; Winter, S. Biochemistry 2005, 44, 8948-8958. [68] Powers, E.T.; Powers, D.L. Biophys. J. 2006, 91, 122-132. [69] Hortschansky, P.; Schroeckh, V.; Christopeit, T.; Zandomeneghi, G.; Fandrich, M. Prot. Sci. 2005, 14, 1753-1759. [70] Hofrichter, J. J. Mol. Biol. 1986, 189, 553–571. [71] Foderà V., Cataldo S., Librizzi F., Pignataro B., Spiccia P., Leone M. , J. Phys. Chem. B, 2009, 113, 10830-10837. [72] Brown, W. Light Scattering, principles and development; Claredon Press: Oxford, 1996.

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In: Protein Aggregation Editor: Douglas A. Stein, pp. 139-159

ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 5

On the Aggregation of Albumin: Influences of the Protein Glycation Philippe Rondeau1*, Giovanna Navarra2, Valeria Militello2+ and Emmanuel Bourdon1 1

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Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Groupe d’Etude sur l’Inflammation Chronique et l’Obésité (GEICO) Université de La Réunion, Saint Denis de La Réunion, France. 2 Università di Palermo, Dipartimento di Scienze Fisiche ed Astronomiche, Via Archirafi 36 90123 Palermo, Italy

Abstract Free radicals are a normal component of cellular oxygen metabolisms in mammals. However, free radical-associated damage is an important factor in many pathological processes. Aggregation, glycation, and oxidative damage cause protein modifications, frequently observed in numerous diseases. Albumin represents the most abundant circulating protein. Many epidemiological studies have established an inverse relationship between the level of serum albumin and the risk of death. Albumin is involved in several biological functions, including the regulation of oncotic pressure, and the binding and transport of many molecules. In addition, albumin displays potent antioxidant and free radical scavenging activities through the redox cycling of its free thiol and its ability to bind metal ions. Albumin constitutes a well-known protein capable of self-assembling in aggregates and also sensitive to glycative modifications, especially *

Corresponding author GEICO, 15 avenue René Cassin BP7151, 97415 Saint-Denis, Réunion, France Tel: (+262) 262 938 648 - Fax: (+262) 262 938 237 Email: [email protected] + Italian National Research Council, Institute of Biophysics at Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy

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Philippe Rondeau, Giovanna Navarra, Valeria Militello et al. in cases of diabetes. In this review, we primarily report the different beneficial activities exerted by albumin, a multifunctional protein. We detail its importance in Human Physiology. Effects of aggregation modifications on albumin’s structure/function relationship are specified, bringing together recent insights on how aggregation processes in albumin can be affected by the protein glycation phenomenon.

Introduction Albumin is one of the longest known proteins since their physiological properties were recognized for the first time by Hippocrates (400 AC) [1]. Albumin is also probably the most studied of all proteins for its manifold functions which constitute the prime interest of scientists from many fields, including biologists, biochemists, chemists, physicians, and also pharmacologists for more than 150 years. Lastly, albumin is the most prominent soluble protein of any species of vertebrates with a concentration between 35 and 50 g/l in plasma [2]. Mainly produced by the liver (99.9 %), albumin is directly discharged in the blood stream where the protein is the shuttling cargo of various endogenous and also exogenous compounds between liver and peripheral tissues.

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Importance of Albumin in Human Physiology Many epidemiological studies have established an inverse relationship between serum albumin levels and mortality risk [3, 4]. In diseased populations as well as in the general population, it has been estimated the odds of death increase by about 50% for each 2.5 g/l decrement in the initial albumin level. The beneficial effects of higher albumin concentrations could be explained by the antioxidative effect of this protein exerted in plasma, a body compartment exposed to continuous oxidative stress [5]. Indeed, owing to its high abundance in plasma, albumin plays a key role in the antioxidant defenses developed by organisms to protect it from oxidative attack [6]. Main specific sites in albumin were depicted in the involvement of this antioxidant activity, such as Cysteine-34, or the more exposed methionine residues [7]. Besides its protective role as a circulating antioxidant, albumin has several other important and pharmacological functions which give the protein an essential position in human physiology. It is a key element in the regulation of osmotic pressure and the distribution of fluids between different compartments. Besides, through its affinity for quite diverse metabolites including metals ions, fatty acids, and drugs (Figure 1), albumin is involved in the improvement of pharmacokinetic profiles of many therapeutic drugs [8].

Structural Aspect of Albumin The many biological functions of this protein could be attributed to its different levels of structure, which are not quite different between each animal species. For instance, the bovine form serum albumins (BSA) has a high polypeptidic sequence homology (76%) with the human form (HSA). Both are the most studied albumins; HSA for its obvious importance in clinical studies, and BSA for its involvement as a model protein in in vitro applications.

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Fiigure 1. Differen nt metabolites traansport pathwayy by albumin betw ween liver and peripheral p tissues. FA: Fatty accid; Trp: Tryptop phan; Cys: Cysteeine; PLP: Myellin proteolipid prrotein.

BSA consttituted by 582 amino acids has h a molecullar weight of 66.28 6 KD [9, 10] and its sttructural shap pe is ellipsoiddal. BSA moolecules, as well w as HSA, are made up u of three hoomologous do omains (I, II, III). In particcular, domainss I and II andd domains II and III are coonnected thro ough helical extensions, e crreating the tw wo longest helices h in albbumin. The seecondary stru ucture of BSA A and HSA is predominnantly alpha-hhelical (67%)) with the reemaining poly ypeptide occuurring in turnns in extendeed or flexiblee regions bettween subdoomains [11]. Seventeen dissulphide bridgges inside the protein makee the tertiary structure s at neeutral pH and d room temperrature stable, but b do not preevent significaant changes inn shape and siize as a functio on of pH and temperature t [111, 12]. A freee thiol group (Cys34) ( is alsoo present in thhe protein strructure; it haas been verifiied that this one has a reelevant role in thermal agggregation patthways of BSA A in appropriaate experimenttal conditions [13]. The album min molecule is i not uniform mly charged within w the prim mary structuree, although thhe charge distrribution on the tertiary struccture seems faairly uniform. Its low isoeleectric point arround 4.9 [14], compared with w physiologgical pH and its low moleccular weight cooperating c foor the mainten nance of oncotic pressure [155]. Moreover, flexibility of o albumin structures alloow the proteein to accom mmodate to m molecules of many m differennt structures [16] [ and give to protein thhe capacity too bind and trransport quite diverse metabbolites. ws several connformational isomerizations i s as a functioon of pH, som me of these BSA show beeing physiolo ogical. Becausse of the greaat number of charged aminno acids in thhe primary seequence of alb bumins, lots of these moddifications must be attributeed to the breaak of ionic coouples caused d by a change of pH value. The three-dim mensional orgganization of the protein chhanges going from the N form (native forrm), in the pH H range 4-8, to the less comppact F form (tthat is a faster migrating forrm that preludee the unfoldinng of the protein), close to pH 4, where a decrease of th he helical struuctures is obseerved . In the pH p region 7-99, albumin unddergoes the

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so called “neutral-to-base” (N-B) transition that is a partial destabilization of the molecule with a loss of rigidity. It involves the interactions between domains I and II, probably due to the disruption of salt bridges at high pH values [22]. Structural and conformational changes of the native structure as a function of pH can also be ascribed to variations in the net charge of the protein [[23-25], as it is positive under the isoelectric point (pH 4.9-5.0) [14, 26]. The net charge of a protein modified through pH values or also by adding metal ions can have implications on the electrostatic interactions between the different domains ,and consequently on the secondary and tertiary structure [24, 27]. BSA and HSA have a tight homology in the primary sequence and structure: although main differences consist of the substitution of two residues having the same hydrophobic nature and therefore not influencing the structure and the protein pI [14, 28, 29, 26]. Unlike BSA, HSA has only one tryptophan (Trp214), localized in domain II [30, 31], which undergoes different pH-dependent structural transitions and conformational changes with different involvements of domains [20] that can bring it to amyloid fibril formation [32].

Albumin Aggregation

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BSA Structural Modifications and Aggregation A crucial relationship lives between conformational and structural properties of proteins and intermolecular interactions, giving rise to the aggregation processes [33-35]. Protein aggregation can certainly be considered one of the most interesting and challenging topics in current research. Such phenomenon is of critical interest today because of its relevance in many fields of scientific and technological research, from medicine to food sciences. Protein molecular assembly is characterized by multiple interactions and cross-feedback among different processes, in particular conformational changes and intermolecular interactions, which strongly affect each other. In addition, a remarkable role may be played by solvent induced/guided interactions. The hierarchy of all these mechanisms and their extent depends on the environment and on several physical and chemical parameters such as temperature, pH, ionic strength, denaturant addition, etc. [36-41]. In general, it is commonly accepted that the aggregation process takes place because of misfolded and partially unfolded states acting in competition with the normal folding pathway [42, 39, 43, 44, 41]. Moreover, we are reminded that in general, the loss of the protein structural stability caused by modifications of pH, temperature, and other external factors may trigger partial unfolding of the native protein, which, in turn, may result in the formation of supramolecular aggregates. Indeed, it is now recognized that in opportune conditions almost all proteins can undergo partial unfolding and aggregation processes. The complexity of the aggregation pathway may give rise to very diverse aggregate morphologies depending on solution conditions, such as oligomers, amorphous structures, protofibrils, mature amyloid fibrils, and gels [45-47, 27, 48]. Both in bovine (BSA) and human (HSA) form, as well as in many other globular proteins, depending on the pH value, albumin has the tendency to aggregate in morphologically different macromolecular assemblies when subjected to a temperature increase [49, 13, 50]. Thermal aggregation of BSA appears to be a multiple interaction and

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cross-feedback among different processes occurring on different length-scales, namely: i) protein conformational changes (possibly including oligomer formation), ii) nucleation and growth of aggregates (fibrils, fibers, gels, amorphous deposits), and iii) the liquid-liquid demixing (LLD) phase transition [51, 52, 47, 27, 53-55]. As suggested by studies on different proteins, protein unfolding and its structural modifications are at the basis of multistage aggregation mechanisms [56, 57]. In thermally induced aggregation, the compact native form of protein, with its well defined secondary and tertiary structures, becomes more flexible on heating and more reactive towards its neighbors. In this regard, the native secondary structure of the protein is strictly related to its aggregation propensity. It has been observed that proteins with richer native β-structures and with greater numbers of hydrophobic residues have a greater tendency toward the creation of aggregates [33, 58, 59]. Nevertheless, the aggregation is not a prerogative of all-β proteins only, but all-α and α+β proteins also create aggregates through β-sheet-like interactions after a conversion of the structure at secondary level from α-type to β-type [60, 61, 13, 47, 62, 27]. BSA molecules, when incubated in mild conditions (1mM, pH ≈ 7, T ≈ 60°C), undergo a partial unfolding that is completed in about 20 minutes [61, 63, 47, 27]. Simultaneously, modifications in the protein secondary structure occur too. As a consequence of heating, the native α-helix structures convert into intermolecular antiparallel β-structures whose appearance indicates formation of BSA aggregates [61, 13, 63, 47, 27]. This trend results in no modifications at changing pH values; however, while the modifications are not relevant between pH 4.5 and 6 [64, 65], the extent of the β-structure formation increases with the moving of pH away from pI [61] and with the increase of the temperature [66, 54]. Studies, carried out at lower concentrations, have allowed the obtainment of information on local modifications of the tertiary structure in the environment surrounding the aromatic residues of BSA, such as the tryptophans [67, 31, 68, 69]. BSA has two tryptophans: Trp-134 located in proximity of the protein surface, but buried in a hydrophobic pocket of domain I, and Trp214, located in an internal part of domain II [31]. Fluorescence measurements have shown that, as a consequence of protein heating, the tryptophans are on average more exposed to the solvent, in agreement with the partial opening undergone by the whole protein molecule [13, 68]. In particular, considering that Trp-134 is more accessible to the solvent than Trp-214 and comparing results obtained for BSA and HSA (having only the Trp-214), modifications in the emission band of tryptophans were assigned principally at Trp-134 allocated in domain I [13, 65]. Thus, as a consequence of heating, BSA molecules undergo partial unfolding and are characterized by the conformational changes of their native structure from a one prevalently constituted by α-helices to more open β-rich structures. These changes are modulated by the pH value, the temperature, the time of heating, and the protein concentration [70, 71, 61, 47, 27, 68, 72, 65]. The conformational changes play a fundamental role in the aggregation pathways. The partial unfolding of the protein causes the exposition of sites, like hydrophobic surfaces or thiol groups (SH), inaccessible in the native form and nevertheless having a dominant role in the aggregation processes. The formation of aggregates via noncovalent interactions and thiol/disulfide exchange reactions may occur simultaneously or sequentially. For example, it is generally accepted that in specific proteins at neutral pH, hydrophobic interactions and thiol/disulfide exchange reactions, leading to the formation of intermolecular disulfide bonds,

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are involved [73-75, 13, 76, 77]. In experimental conditions appropriate for the growth of BSA aggregates, the aggregation can proceed via hydrophobic interaction and/or via thiol/disulfide exchange reactions. The pKa of a typical thiol group is roughly 8.3, but can vary due to its environment [78]. As a consequence, the reaction efficiency and the resultant aggregation pathway are modulated by pH [61]. Indeed, at pH far from the pKa of the thiol/disulfide exchange reaction, the reaction may occur with low probability, and the hydrophobic interactions play a crucial role in the formation of aggregates. However, it is also important to stress that the net charge of the protein can play a key role in the undertaking of specific aggregation pathways. The protein net charge can be easily altered, changing the pH values in respect to the protein pI. At extreme pH values, far from the isoelectric point, the native protein structure is destabilized [23-25] and, moreover, the electrostatic repulsion between protein molecules increases, resulting in a larger tendency to unfold and modify the intermolecular interactions [79, 80]. For all these considerations, the excursus on BSA aggregation is presented here and discussed at the changing of the pH. At pH quite close to the pI (i.e. with a minor net charge in the protein surface), thermally treated BSA form a few small oligomeric structures and a large fraction of big disordered aggregates characterized by an increased amount of random coil structures, as observed by Dynamic light scattering and FTIR measurements [61]. Moreover, studies carried out using an extrinsic dye (anilinonaphtalene-8-sulfonate, ANS) have shown that, in these experimental conditions, the aggregation is driven by strengthened hydrophobic interactions, due to their entropic origin [65, 81], prevailing on the low electrostatic interactions, and on the not promoted exchange reaction. It is important to stress that the comparison between BSA and HSA carried out at pH 4.7 and 5.7 have shown that conformational changes involve only tertiary structures and the aggregation proceeds via disordered and non-specific interactions without relevant conformational changes at the secondary structure for both the proteins, but data concerning HSA provides information on different aggregation pathways with respect to those of BSA [65]. At pH values far from the pI of the protein, i.e. when the net charge of the protein is increased, the aggregation mechanism proceeds in an ordered and slower way to allow the forming of β-aggregate structures that prevalently come from the conversion of α-helix ones and lead to the production of aggregates of small dimension: oligomers [61, 27]. Moreover, at neutral and alkaline pH values, the thiol/disulfide exchange reaction is allowed and it contributes to the aggregate growth. Indeed, it is well known that the unique free cysteine in BSA (Cys-34) has a crucial role in the formation of aggregates [13, 82]. Studies carried out covalently “capping” Cys-34 by Fluorescein-5-Maleimide showed that the aggregation of BSA at pH 6.2 is slower and proceeds involving conformational changes at the tertiary structure level [13]. Fibrils were observed at neutral and acidic pH values in extreme experimental conditions of elevated temperatures and long day incubations, characterized by using specific novel processes [83, 84]. The reaction leading to BSA fibrillation proceeds without a lag phase and it is linearly dependent on the protein concentration. The ease with which BSA forms fibrils highlights once again the ubiquitous nature of fibrillation, which does not exempt even multi-domain all-α proteins; at the same time, it serves to illustrate that fibrils can tolerate a high amount of non-β structure, presumably due to their ability to relegate α-helix structure to parts outside the fibrillar core [83]. Also HSA, strictly homologues to BSA, forms amyloid fibrils in appropriate and several experimental conditions

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[332, 85]. Thesee fibrils havee the structuraal characteristtics of amyloids, as probedd by x-ray diiffraction patterns, affinity to Congo redd and thioflaviin T, birefringgence, and higgh stability. The formation of protofibrilss, curly fibers,, and mature fibrils f by HSA A proteins werre observed byy Juarez et al. 2009. HSA fibrillation f exxtended over several s days of o incubation without w the prresence of a lag l phase, exccept for HSA A samples incuubated at aciddic pH valuess and room teemperature in the absence of o electrolytess. The absencee of a lag phaase occurred iff the initial agggregation waas a downhilll process thatt did not requuire a highly organized annd unstable nuucleus. The fiibrillation proccess was accoompanied, as usual, u by a proogressive incrrease in the β--sheet and un nordered confo formation at thhe expense off α-helical coonformation [557, 85-87]. These changess also involvved the preseence of diffeerent structuraal intermediaates in the a oligomeric clusters (globbules), bead-like structuress, and ringagggregation pathway, such as shhaped aggregaates. The authhors suggestedd that fibril foormations mayy take place through t the roole of associattion-competennt oligomeric intermediates, i , resulting in a kinetic pathw way via the cllustering of these oligomeric species to yiield protofibrils and then fibbrils [85]. The differeent stability off the albumin at a changing pH H values discuussed before iss shown by sccanning T mad de at different values of pH (Figure 2).

Fiigure 2. Static sccattering intensitty (incident lightt λ= 633 nm) as a function of tem mperature for BS SA sample (00.5mg/ml) at diffferent pH: pH 4.7 (z), pH 5.7 ( ), pH 6.2 ( ) and a pH 8.9 ({). The scan rate is 14°C/h uppwards.

For each sample above, a critical tem mperature andd an increase of o scattering intensity i is obbserved. This indicates thaat at temperattures beyond the critical temperature, thhe thermal unnfolding transsition is accompanied by an a irreversiblee aggregationn at temperatuures higher thhan 55°C. Agg gregation proccesses are fasteer at lower pH H towards the protein isoeleectric point, inn agreement with w the conssiderations aboove discussedd with respect to the balannce among ellectrostatic, hy ydrophobic, and a thiol/disuulfide exchangge reactions. Obviously, O thhe different naative conform mational isomeerisation of allbumin dependdent on pH has a relevant role in the diifferent stabiliity of the proteein to thermal stresses [12, 20-22]. 2

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Finally, the important role of the protein concentration must not be neglected in the aggregation processes. It determines the probability for the protein molecules to interact at short-range. Moreover, at fixed temperatures and pH values, the size of aggregates depends on the concentration [88]; in particular, through changing the concentration, it is possible to drive the aggregate’s formation from oligomers to heat- and cold-induced gels [53]. In fact, albumin is a globular protein able to form gels under appropriate conditions. Thermal gelation is commonly seen as a three-step process that can be intertwined: i) first, the native protein unfolds, exposing hydrophobic amino acid residues, favouring in this way the building of molecular pre-aggregates via disulfide bridges, hydrogen bonds, and hydrophobic and/or Van der Waals interactions, ii) then, the aggregation further proceeds with the association of protein pre-aggregates, and iii) finally, when the protein concentration is sufficiently high, a three-dimensional network entrapping water is created [89]. Once again, pH values and electrostatic interactions have a crucial role in determining the gel structure and its macroscopic properties, such as gel strength, elasticity, and water holding capacity [90]. Depending on the pH value and ionic strength of the medium, different kinds of protein gels have been obtained, varying from transparent to turbid and opalescent “fine stranded” or “particulate” gels. At pH values around the protein isoelectric point, pI, and high extrinsic ionic strengths, the resultant gel networks are heterogeneous and largely opaque; they are characterized by a random association with large and almost spherical aggregates and are named “particulate gels” [89, 91]. These originate from a faster protein aggregation which does not allow the necessary structural reorganization to enable the formation of more ordered structures [85, 89]. At pH values far from the isoelectric pI, fine but highly persistent fibrillar networks are formed rather than particulate stranded gels [89, 90, 92, 93]. They are composed of more or less flexible linear strands and for this reason are named “fine stranded” gels. In particular, both heat- and cold-gels can form when albumin concentration is above 2.5 % (v/v) [47, 72, 94-97]. The characteristic evolution of the aggregation process of albumin and the macroscopic properties of the cold gels of this protein are affected by the presence of salts or metal ions in the solution [27, 96, 98]. Indeed, BSA is able to form specific bonds with metal ions; at least two metal binding sites have been identified in hydrophobic cavities of the molecule [99102]. Divalent metals can play a promoting role in protein aggregation thanks to their ability in acting as bridges, as well as changing the ionic strength of the solution by providing an electrostatic screening between the negatively charged groups of the neighboring protein molecules [47, 90]. The loss of repulsive forces allows the charged protein molecules to get close enough to interact via noncovalent forces with a low potential energy [90]. The effect of copper or zinc on the evolution of BSA aggregation has been studied [27]. Acting as a bridge between protein molecules, zinc, more than copper, promotes aggregate growth; this different effect is also due to their different affinities and structural coordination to the protein. On the contrary, the propensity of Cu(II) ions undergo intramolecular chelation explains the absence of a relevant effect on protein aggregation, although it induces structural changes in the protein [27].

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Albumin Aggregation – Consequences of the Protein Glycation

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Albumin Glycation Impact on Albumin Structure One feature of such plasmatic protein modifications is the effect of nonenzymatic glycation of circulating albumin which can occur in cases of hyperglycaemia or diabetes. This process, also known as Maillard reactions, is a slow non-enzymatic reaction that initially involves the attachment of glucose or derivatives with free amine groups of albumin (lysine or arginine) or free cysteine, to reversibly form a Schiff base product, leading to a formation of stable fructosamine residue (ketoamine) after Amadori rearrangement [103, 104]. The Amadori products could subsequently cyclize, forming pyranose or furanose carbohydrate adducts [105]. Further modifications of these early stage glycation products, such as rearrangement, oxidation, polymerization, and cleavage, give rise to irreversible conjugates named advanced glycation end products (AGE) [105]. In vivo, the proportion of glycated albumin in healthy persons is in the range of 1% and 10% [28, 106], and in the case of diabetes mellitus, it could increase two-to threefold [107]. This glycated albumin rate could even reach more than 90% for severe diabetic patients with poor diabetic control [108]. Glycation processes affect plasmatic proteins, such as haemoglobins or immunoglobulins, and proteins from the extracellular matrix with long lifespans, including collagen, laminin, and fibronectin. Owing to its long half-life time (21 days) and its high concentration in plasma, serum albumin is a circulating protein particularly sensitive to glycation. The main studies on the structural, biological, and physiological characterizations of glycated albumin were performed using in vitro or in vivo models of glycation derived from human albumin and bovine albumin, as well. Glycoxidation of albumin is associated with important structural modifications. Among these modifications, several studies have reported the quenching of tryptophan fluorescence in modified albumin upon glycation reflecting a local unfolding around these residues [68, 107, 109]. This structural modification was accompanied by a partial opening of hydrophobic pockets for albumins (BSA and HSA) glycated in vitro for 21 days with glucose [68, 110]. Indeed, a stronger affinity of these modified proteins for anilinonaphtalene-8sulfonate (ANS) probes was observed for a short incubation time of protein with glucose (below 21 days). This growing affinity could be attributed to a higher exposure of hydrophobic sites to the solvent [111]. In contrary, a longer incubation (more than 21 days) of albumin with glucose tends to inhibit penetration of ANS into hydrophobic domains of the protein [110, 112]. The decrease in hydrophobicity of the long term modified albumin is explained by the formation of the molten globule-like state and the amyloid nanofibril after 3 and 20 weeks of incubation with glucose, respectively [110, 113].

Albumin Glycation is Characterized by Aggregates Growth Several works suggested narrow links between protein aggregation and glycation [113, 114]. For instance, multimerization or condensation of glycated proteins into plaque is Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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already observed for extracellular matrix proteins such as fibrinogen or collagen, and also for circulating proteins including albumin [115]. On Figure 3 of this chapter, an enhanced formation of aggregates is observed when albumin is glycated by glucose or methylglyoxal (MGO) incubations. Besides, amyloid type lesions and glycated proteins share common properties. The receptors for advanced glycation end products (RAGE) is thought to be a primary transporter for amyloid type peptides and terminal products of glycation [116]. Other scavenger receptors such as MSR-A, MSR-B, and CD36 are likely to bind to AGE as well as to amyloid fibrils [117]. At last, the involvement of AGE is well known in the progression of many neurodegenerative diseases including Alzheimer’s. In this pathology, advanced glycation end products are identified to promote additional amyloid deposits [118].

Figure 3. Glycation effects on albumin aggregation. Electrophoretic migration profiles (SDS-PAGE) of non glycated BSA and glycated BSA with 10 mM methylglyoxal (BSAMGO) for 2 days and with 100 mM glucose (BSAG100) for three weeks at 37°C.

Regarding albumin, alterations of its tertiary structure with glycation processes generally does not impact the secondary structure of the protein systematically and significantly , as it was also probed after glycation for seven long weeks by FTIR absorption and circular dichroism [119, 68]. However, a prolonged incubation (more than seven weeks) with carbohydrates (glucose, fructose, or ribose) induces a transition from a helical structure to a β-sheet of albumin, which is the base amyloid formation [113]. In this last study, Sattarahmady et al. reported three different structural organizations for HSA incubated for a long time with glucose: large branched chains of globular aggregates, bundles of unbranched fibrillar aggregates and fine amorphous aggregates. Besides, the nature of carbohydrate attached to the protein impacts on appearances of amyloid structures. Indeed, incubation of HSA with ribose leads to formations of several forms of aggregates, such as long straight amyloid fibrils and fibrous sheet-like structures, drastically different to those obtained from HSA with glucose [113]. Other studies have reported conversions of helicoidal secondary

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structures into β-sheet forms in albumin resulting from glycation processes. According to Khan et al., glycation of albumin could generate thermodynamically more stable high molecular weight aggregates with a high content of β-sheet structures compared with its non glycated form [120]. This conversion of secondary structures of albumin into β-structures is supported by the fact that both glycated ligands derived from albumin and amyloid ligands can bind to the same multiligand receptors, named t-PA, and respond to the regulation in blood fibrinolysis [121]. Bouma et al. have also investigated the role of glycation in amyloid formations. Firstly, glycation of bovine serum albumin with glucose-6-phosphate (G6P) leads to formations of dimers and multimers consisting of intermolecular cross-links, such as dialkyldihydropyrazine lysine-lysine cross-links [122]. Moreover, they show different structural organizations of aggregates according to the time of incubation of albumin with G6P. Indeed, the two week glycation period induced formations of bundles of unbranched fibrillar aggregates while a longer incubation yielded amorphous aggregates (4 weeks) or fibrous sheet-like structures after 23 weeks. At last and similarly to Sattarahmady’s work, Bouma et al. showed that albumin forms amyloid irrespective to glycating agents. Indeed, whatever the nature of glycating agents (glucose, fructose, g6p, glyceraldehyde…), crosslinking following AGE formation may be at the root of amyloid formation. According to Bouma, conversion of globular native albumin to the amyloid cross β-structure depends on the nature of AGE products rather than carbohydrate adducts. However, aggregate formation induced by glycation is not always associated with secondary structure modification. For instance, short time incubation (7 days) with D-ribose causes albumin to misfold rapidly and form globular amyloid-like aggregations without any changes in α-helix/ β-sheet proportions [123]. In this study, Wei et al. showed first that furanose type sugar such as D-ribose reacts much more rapidly with albumin in the formation of glycated products. Secondly, in such glycation processes, conversions of α-helix structurse into β-sheets is not a prerequisite for globular amyloid-like aggregation of ribose-glycated BSA. Finally, these amyloid aggregates formed by glycation are involved in apoptosis in the neurotypic cell line. Even if Stefani & Dobson have investigated the toxicity of globule-like protein aggregates in neuron cells [59], the origin of such glycated products should be clarified. Indeed, toxicity of glycated albumin may be due to modifications of protein into AGE, bringint it to its aggregative state. However, the mechanism underlying glycation–induced amyloid formation must also be clarified. Indeed, conversion of globular states of native proteins into a fibrillar state from glycation could be attributed to a modification of amino groups at the solvent exposed site of polypeptides which stimulate refolding. Bouma et al. suggest two ways of modification: 1) local or global unfolding may be induced by mechanical stress on the polypeptide exerted by intermolecular or intramolecular AGE-bridged crosslink, and 2) the polypeptide chain could be partially unfolded due to an alteration of the microenvironment of lysine or arginine residues in the presence of carbohydrate adducts. Upon glycation, particular parts of polypeptide sequences initially hidden in globular states may become more exposed to solvent. This unfolding of glycated albumin facilitates new contacts between amino-acids residues and stimulates formations of cross-β-structures, a prerequisite for amyloid fibril formation.

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G Glycated Alb bumin Show wed Lower Propensity P For Therma al Aggregattion

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n processes arre thought to be involved in i the converssion of globullar albumin If glycation innto amyloid ty ype aggregatess, it could alsoo have a deterrminaning rolee in the inhibition of this agggregation pro ocess when innduced by heaating treatmentt. Several studdies investigatted thermal agggregation pro ocesses at 58°°C of BSA, innitially glycatted with differrent amounts of glucose foor different tim mes of incubaation [68, 124]]. In these studdies, a similarr partial unfolding of the teertiary structu ure which acccompanies thhe thermal agggregation proocess regardlless of the gllycation exten nt of BSA wass observed. Onn the contrary, the conversioon of similar amounts a of α-helix structu ures is distribuuted differentlly between inntra and interm molecular β-ssheets. The u for glyccation was laatter progressively decreassed when thee glucose cooncentration used inncreased [68]. In parallel, thhe formation of o aggregates is inhibited with w increasingg glycation exxtent of BSA A induced by increasing thhe time of inncubation (Figgure 4a) or by b growing cooncentrations of glucose (Fiigure 4b). Theerefore, the agggregation proccess of BSA is markedly afffected by the glycation. Seeveral explanaations could bee suggested foor this low susceptibility off aggregation for highly gllycated album min. First of alll, it has alreaady been show wn that the unnique free cyssteine of BSA A, analogouslyy to HSA, has a noteworthyy role contribuuting to the agggregate’s gro owth by the foormation of inntermolecular disulphide d briidges and S-S exchanges [113, 82]. The involvement of the uniquue, free cysteine in aggreggation pathwaays can be afffected for gly ycated albuminn. Indeed, nonn-enzymatic gllycation of prooteins generally involves am mino groups (lysine ( and argginine residuees) [125], and also thiol grooups of cysteiine residue, duue to its poweerful nucleophhile property [1126, 127]. In the t case of glyycation, the addduction of caarbohydrates on o the uniquee sulphydryl group g could occur, o preventting the cysteiine residue frrom taking parrt in the formaation of interm molecular bondds.

Fiigure 4. a) Time evolution of thee Rayleigh peak intensity @ 2700 nm for 1:50 diluted BSA (20mgg/ml) in phhosphate buffer 0.1M 0 pH 7.4 whhen heated at 65 °C after incubattion with 200 mM M Glucose (18m mg/100ml) at 377°C for 0 day ( ), 30 days ( ), 50 5 days (z), 70 days ({). b) Electrophoretic miigration profiles (nativePA AGE) of non gly ycated BSA and glycated BSA with w 10 mM methhylglyoxal (BSA AMGO) for 2 dayss and with 1000 mM glucose (BSA ( bated at 58°C forr 250 minutes. (11), (2) and (3) inndicate monomerrs, dimmers G100) incub annd oligomers ban nds of BSA, resppectively.

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Besides, due to the glycation process, the tertiary structure of modified albumin is not similarly altered via the aggregation pathway. Indeed, tertiary conformational changes occur upon glycation as a consequence of molecular rearrangement after the formation of AGE products. In contrary to aggregation, glycation processes of albumin is associated with an increase in its hydrophobicity. This specific misfolding of albumin due to glycation could also contribute to reducing its sensitivity to form aggregates.

Conclusion

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Aggregation and glycation processes induce protein modifications, and these phenomena have increasingly been involved in disease progression. Albumin not only represents the most abundant protein in plasma, it also constitutes the most studied protein. Even so, when discussing the importance of the protein, most underestimate the impact of protein modification, arguing against the high quantity of this ‘‘sponge’’, which binds all kinds of molecules in the circulation. The quantity of albumin certainly constitutes a determinaning health/nutritional indicator: the odds of death increase by about 50% for each 2.5 g/l decrement in the initial albumin level. Enhanced aggregation of albumin as well as its glycation status exerts a dramatic impact on the protein structure. In summary, glycated albumin is characterized by aggregate formations and exhibits a reduced propensity for thermal aggregation. Additional studies remain highly warranted in order to achieve a better understanding of the influence of albumin glycation on its propensity to undergo aggregation phenomenona.

Acknowledgments We are grateful to all members of the MBSM group (http://www. fisica.unipa.it/ biophysmol/) for continuous stimulating discussions, and a particular acknowledgment goes to Valeria Vetri for her contribution in the scattering measurements. This work was supported by the Ministère de l’Enseignement Supérieur et de la Recherche et de l’Outre Mer, le Conseil Régional de La Réunion, the Université de La Réunion.

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Philippe Rondeau, Giovanna Navarra, Valeria Militello et al. Halliwell, B. (1988). Albumin--an important extracellular antioxidant? Biochem Pharmacol, 37, 569-71. Halliwell, B. (1996). Antioxidants in human health and disease. Annu Rev Nutr, 16, 3350. Roche, M.,Rondeau, P.,Singh, N.R.,Tarnus, E. & Bourdon, E. (2008). The antioxidant properties of serum albumin. FEBS Lett, 582, 1783-1787. Kratz, F. (2008). Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release, 132, 171-83. Gelamo, E.L. & Tabak, M. (2000). Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants. Spectrochim Acta A Mol Biomol Spectrosc, 56A, 2255-71. Peters, T., Jr. (1985). Serum albumin. Adv Protein Chem, 37, 161-245. Carter, D.C. & Ho, J.X. (1994). Structure of serum albumin. Adv Protein Chem, 45, 153-203. Foster, J.F. (1977). Albumin Structure, Function and Uses Oxford: Pergamon. Militello, V.,Vetri, V. & Leone, M. (2003). Conformational changes involved in thermal aggregation processes of bovine serum albumin. Biophys Chem, 105, 133-41. Brewer, S.H.,Glomm, W.R.,Johnson, M.C.,Knag, M.K. & Franzen, S. (2005). Probing BSA binding to citrate-coated gold nanoparticles and surfaces. Langmuir, 21, 9303-7. Figge, J.,Rossing, T.H. & Fencl, V. (1991). The role of serum proteins in acid-base equilibria. J Lab Clin Med, 117, 453-67. Weber, G. (1975). Energetics of ligand binding to proteins. Adv Protein Chem, 29, 183. Katchalski, E.,Benjamin, G. & Gross, V. (1957). The Availability of the Disulfide Bonds of Human and Bovine Serum Albumin and of Bovine γ-Globulin to Reduction by Thioglycolic Acid. Journal of the American Chemical Society, 79, 4096-4099. Nakamura, K.,Nakazawa, Y. & Ienaga, K. (1997). Acid-stable fluorescent advanced glycation end products: vesperlysines A, B, and C are formed as crosslinked products in the Maillard reaction between lysine or proteins with glucose. Biochem Biophys Res Comm 6, 227–230. Sadler, P.J. & Tucker, A. (1993). pH-induced structural transitions of bovine serum albumin. Histidine pKa values and unfolding of the N-terminus during the N to F transition. Eur J Biochem, 212, 811-7. Dockal, M.,Carter, D.C. & Ruker, F. (2000). Conformational transitions of the three recombinant domains of human serum albumin depending on pH. J Biol Chem, 275, 3042-50. Kosa, T.,Maruyama, T.,Sakai, N.,Yonemura, N.,Yahara, S. & Otagiri, M. (1998). Species differences of serum albumins: III. Analysis of structural characteristics and ligand binding properties during N-B transitions. Pharm Res, 15, 592-8. Harmsen, B.J.,De Bruin, S.H.,Janssen, L.H.,Rodrigues de Miranda, J.F. & Van Os, G.A. (1971). pK change of imidazole groups in bovine serum albumin due to the conformational change at neutral pH. Biochemistry, 10, 3217-21. Bendedouch, D. & Chen, S. (2002). Structure and interparticle interactions of bovine serum albumin in solution studied by small-angle neutron scattering. The Journal of Physical Chemistry, 87, 1473-1477.

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spectroscopy and PAGE: relations between structural changes and antioxidant properties. Arch Biochem Biophys, 460, 141-50. [125] Day, J.F.,Thorpe, S.R. & Baynes, J.W. (1979). Nonenzymatically glucosylated albumin. In vitro preparation and isolation from normal human serum. J Biol Chem, 254, 595-7. [126] Thorpe, S.R. & Baynes, J.W. (2003). Maillard reaction products in tissue proteins: new products and new perspectives. Amino Acids, 25, 275-81. [127] Zeng, J. & Davies, M.J. (2005). Evidence for the formation of adducts and S(carboxymethyl)cysteine on reaction of alpha-dicarbonyl compounds with thiol groups on amino acids, peptides, and proteins. Chem Res Toxicol, 18, 1232-41.

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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

In: Protein Aggregation Editor: Douglas A. Stein, pp. 161-178

ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 6

The Role of Conformational Domain Lability of Fibrinogen Molecules in Processes of Self-Assembly of Fibrin Monomers and Fibrinogen Aggregation M. A. Rosenfeld, V. B. Leonova and M. I. Biryukova

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Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina 4, Moscow, 119334 Russia

Abstract Fibrinogen is the major plasma protein of the blood coagulation system, molecular domain structure of which strictly corresponds to its main function – formation of insoluble fibrin. The mechanism of binding sites interaction localized on fibrinogen domains in order to construct protofibrils and fibrils proves to be well known. However, the role of conformational domain lability of fibrinogen molecules in processes of self-assembly of fibrin monomer and fibrinogen aggregation is still far from being completely understood. The article summarizes the data regarding fibrinogen domain D as being the most capable to local conformational rearrangements. Three-dimensional organization of intermediate soluble forms of fibrin-polymers in the presence of non-denaturizing urea concentration has been studied. Using the methods of dynamic and elastic light scattering combined with the analytic ultracentrifugation and viscosimetry it was shown that along with formation of traditional double-stranded protofibrils, in which fibrin monomer molecules by virtue of interactions between outer domain D and central domain E were arranged in a staggered overlapping manner, there was an alternative route of generation of equilibrium abnormal single-stranded, rod-like protofibrils formed in “end-to-end” fashion. It was concluded that local conformational transformation in regions of domains D remained the only way of the “end-to-end” association. It was suggested that fibrinogen molecules could undergo local conformational transformation in regions of fibrinogen domains D during their incubation in a solution

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M. A. Rosenfeld, V. B. Leonova and M. I. Biryukova under the conditions close to the physiological ones. This results in accumulation of socalled “defective” molecules responsible for the process of fibrinogen aggregation. Molecules interact in “tail to tail” manner with formation of flexible single-stranded chain polymers. On attaining a critical length, polymers twist into a coil and aggregate to form branched clusters in which the segments are packed sufficiently dense to resemble strongly hydrated globular particles. It was suggested that damage of a native threedimension organization of domains D causes an opening of the new reaction sites, which largely differed from the polymerization sites a and b “holes”. The effect of molecular “aging” of fibrinogen stimulated by pre-incubation in a solution both on the fibrin architecture and its ability to cross-link under the action of factor XIIIa has been studied. Fibrin generated from “defective” fibrinogen molecules had a coarser structure to be characterized by a higher mean mass-length ratio of the polymeric fibers compared to the native fibrinogen. Our data indicate that structurally modified fibrinogen molecules had more affinity for interchain cross-linking by formation of the ε/(γ-glu)lys isopeptide covalent bonds. The physicochemical mechanism of the fibrinogen molecular aging was demonstrated to be determined by processes that were identical to either its spontaneous oxidation (during incubation of this protein in a solution) or induced oxidation. Data on IR- and EPR-spectroscopy methods obtained for oxidized fibrinogen fragments D and E make us believe the peripheral D-domains of fibrinogen as being the most sensitive to the free-radical oxidation. It is followed by the local structural conversions in regions of the domain D to expose reaction centers responsible for interaction in “end to end” fashion. The conjectural mechanism of conformational fibrinogen instability is discussed.

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Keywords: fibrinogen, fibrin, self-assembly, aggregation, domains D and E, conformational instability.

Introduction Fibrinogen is the key plasma protein in the blood, clotting molecular structure of which strictly corresponds to its main function – formation of the insoluble fibrin. The conversion process of the fibrinogen to the fibrin was being investigated since 1940s. Today’s concepts of the fibrinogen and fibrin structure and properties have been developing as the experimental and theoretical data continued to accumulate. The size and the shape of fibrinogen and the morphology of fibrin were first studied in parallel investigations by the classical methods of hydrodynamics and electron microscopy. However, subsequently the hydrodynamic methods were replaced by the electron microscopy and supplemented by the different types of light scattering, chromatography, chemical, and enzymatic fragmentation. It is now well known that the fibrinogen molecule has a complex dimeric structure consisting of three pairs of polypeptide chains: (Aα, Bβ, γ)2 [1]. The Aα-, Bβ.-, and γ-chains were shown to consist of 610, 461 and 411 amino acid residues [2], with their molecular weights calculated from the amino acid sequences being 67, 55 and 47.5 kDa respectively. The half-molecules are covalently linked through symmetrical disulfide bonds at the Nterminal ends. The central part of the molecule, which comprises the NH2-terminal regions of all polypeptide chains, forms the central E domain containing two polymerization centers (A and B “knobs”) shielded by fibrin peptides A and B. E domain. This is linked via coiled-coil structures to two peripheral D domains that have open polymerization centers (a and b

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“holes”) complementary to the E domain centers. Furthermore the COOH-terminal regions of the Aα-chains are packed into the αC domains, which appear to interact with each other near to the central E domain [3]. The monomer fibrin is formed from fibrinogen via thrombin-induced removal of the A and B fibrin peptides from the Aα- and Bβ-chains, which exposes A and B “knobs” (polymerization centers). The “knobs” are complementary to a and b “holes” located respectively in γC- and βC-subdomains in the COOH-terminal regions of the D domain. The N-terminal “knobs” of the α-chains in one molecule of the monomer fibrin bind to the “holes” of the receptor a in the terminal γC-subdomains of the adjacent molecules, leading to the formation of the staggered overlapping rigid rod-like double-stranded fibrin protofibrils. The N-terminal “knobs” of the β-chains interact with receptor b “holes” in the βC-subdomains promoting assembly of double-stranded fibrin protofibrils [4, 5]. In addition the fibrin peptides having been released from the Aα- and Bβ-chains fibrinogen under the action of the thrombin, the αC domains are likely to be dissociated from the central E region of the protein. Thus the αC domains can take place in the conversion of protofibrils into fibrils [6, 7]. Subsequently, the fibrils undergo lateral associations to create the multi-stranded fibers [8, 9]. Two types of branch junctions are known to occur in the fibrin networks [10]. The first type gives rise to lateral interactions of a double-stranded fibril with another fibril to form a fourstranded fibril, a so-called “bilateral” junction. The second type of branch junction, termed “equilateral”, is driven by convergent interactions among the three fibrin molecules leading to the generation of the three double-stranded fibrils [1]. The fibrin polymers undergo secondary transformation through fibrin-stabilizing factor XIIIa. Cross-linking of fibrin involves both γ-chains and α-chains by means of the formation of the covalent bonds between glutamine and lysine residues in the fibrin [11]. The COOHterminal regions of γ-chains of contacting D domains are responsible for the formation of intermolecular γ-dimers with a molecular weight of 95 kDa. α-Сhains of each molecule interact with α-chains of two other molecules, which leads to the formation of α-polymers with a molecular weight over 500 kDa [1, 2]. These structures in the fibrin were believed to enhance the stabilization of fibrin at inter-fiber contacts or at branch points, thus increasing the mechanical strength and elasticity of the fibrin.

The Self-Assembly of the Single-Stranded Fibrin Protofibrils As it appears from the above the process of fibrin formation from the fibrinogen involves several stages, most of which have been well studied. The self-assembly of fibrin monomers gives rise to the generation of the double-stranded protofibrils, in which fibrin monomer molecules by virtue of interactions between the outer domain D and the central domain E are arranged in a staggered overlapping manner. It is the key reaction in the formation of threedimensional fibrin network because the self-assembly of rod-like double-stranded protofibrils reflects the unique multi-domain fibrinogen structure. However, it was shown that along with the formation of the traditional double-stranded protofibrils, there was an alternative way to achieve the equilibrium abnormal singlestranded, rod-like protofibrils formed in the “end-to-end” fashion [12, 13]. Methods of the

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dynamic and elastic light scattering combined with the analytic ultracentrifugation and viscosimetry were used to prove a possibility to generate such intermediate soluble forms of fibrin polymers in the presence of non-denaturizing urea concentration. The reactions of the fibrin formation in a medium of various non-denaturing urea concentrations made it possible to obtain equilibrium fibrin polymers and to investigate certain hydrodynamic and optical properties of such a system. It is known that hydrodynamic methods do not permit a strict evaluation of the three-dimensional organization of the protein molecules, which differ substantially in the shape from ellipsoid of rotation, cylinder, or globular structure. According to the generally accepted data, fibrinogen is a rod- or an ellipsoid-shaped particle, having a length of 45 nm and degree of asymmetry of 5 and containing as much as 5-6 g of water per gram of protein [2]. On the basis of this model the fine structural details the asymmetrical molecule could be excluded from consideration. The use of the Yamakawa hydrodynamic theory permitted an evaluation of the influence of the concentration effect on the coefficient of translational friction in a first approximation, liner with respect to the concentration. By using the Svedberg and Kuhn-Mark equations, dependences of the hydrodynamic constants on the molecular masses of the fibrin polymers were constructed. This made it possible to demonstrate for the first time the existence of equilibrium single-stranded, rod-like protofibrils.

From Rosenfeld et al., [12]. Figure 1. Sedimentograms of fibrinogen, fibrin monomer, and various intermediate fibrin polymers. 1) fibrinogen in 3.5 M urea; 2) fibrin monomer in 3.5 M urea; 3-5) fibrin polymers at a urea concentration of 2.8, 2.5, and 2.3 M, respectively. Time of separation for fibrinogen and fibrin monomer is 25 min, for fibrin polymers – 10 min.

The results of the ultracentrifugation of an intermediate fibrin polymer are presented in Figure 1. It is obvious that when the concentration of urea is lowered, the peak corresponding to slowly sedimentary fibrin polymers decrease in amplitude is broadened at the base. This Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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explains an increase in the polydispersion of the sample. In the region of 2.5 M urea, a transition from unimodal to bimodal distribution of the particles is observed according to the sedimentation rate. It points out to an availability of the two different three-dimensional fibrin polymer structures. Polymers composing the slow sedimentary fraction proved to be the single-stranded, rod-like protofibrils. In region 2.5-2.3 M urea both single-stranded, rod-like protofibrils and more compact double-stranded, rod-like protofibrils are simultaneously presented. A strict conclusion on the mechanism of fibrin monomer self-assembly was drawn on the α

basis of the well known Kuhn-Mark equation: [η] = [η] = Kη M S, D , where [η] is the intrinsic

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viscosity, M is the average molecular mass of a polymer calculated according to the values of the Z-average diffusion coefficient and average sedimentation constant by the Svedberg formula, Kη and α are constants in the homologous series of polymers. On curve, as it can be seen from Figure 2, there are two portions differing in slope with α1=1. 3 and α2 = 0.7. It was not difficult to show that the initial portion of the curve corresponds to the formation of single-stranded, rod-like protofibrils, constructed by association of fibrin monomer molecules in “end-to-end” fashion. With decreasing urea concentration the protofibrils grow and when they reach a critical length they form more compact structures as evidenced by the decrease in α. On the basis of the hydrodynamic data, appearance of such structures are given rise to the lateral contacts between rigid rod-shaped protofibrils and as a reason of these events generation of double-stranded, rod-like protofibrils was expected.

Figure 2. The effect of the average molecular mass polymer on the intrinsic viscosity.

Elastic light scattering was applied to investigate the architecture of the soluble equilibrium oligomers at different stages of self-assembly. Scatter angle changes at different light intensity by soluble equilibrium oligomers are illustrated in Figure 3. The tangent of the Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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angle of slope of the asymptotic branch for each curve on the plot can be used to obtain the average fiber mass-length ratio Mω /Lω [13]. If the urea concentration is 2.8 M the average fiber mass-length ratio is 0.61×1011g/mole×cm. For example, if the mass-length ratio of fibrinogen molecule is (to be) 0.63×1011g/mole×cm one may come to a conclusion that under these conditions the oligomers are relatively short single-stranded, rod-like protofibrils. This conclusion is in a good agreement with the results obtained on the basis of hydrodynamic parameters. If the urea concentration decreases to 2.3 M the ratio Mω /Lω of soluble oligomers increases to reach the value of 0.90×1011g/mole×cm. The increase in the average fiber masslength ratio is evidence that more compact structures are appearing in the solution. Since Mω /Lω = (Mi/Li)ωi/ω + (Mk/Lk)ωk/ω, where Mi/Li and Mk/Lk are an average fiber mass-length ratio for less and more compact structures, ω is a total mass concentration of a protein, ωi and ωk correspond to mass concentration of the slow and fast sedimentary fractions (Figure 1), it was possible to calculate the ratio Mk/Lk for compact fast (rapidly) sedimentary polymers. In all cases regardless the urea concentration this value was (1.2-1.3) ×1011g/mole×cm. Thus, the data shows the existence of double-stranded, rod-like protofibrils which are able to be formed either by dimerization of single-stranded protofibrils or by successive addition of monomer molecules in “end-to-middle” type.

Figure 3. The intensity of light scattering by soluble equilibrium oligomers and the scatter angle at different urea concentration. 1) 3.5 M (0.63 ± 0.05), 2) 2.8 M (0.61± 0.06±), 3) 2.5 M (0.71±0.06), 4) 2.3 M (0,90±0.05). Values of Mω /Lω×1011g/mole×cm, indicated in brackets.

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The result obtained from (on) the generation of thermodynamically stable soluble polymer chains constructed by an association of monomer molecules “end-to-end” in the inhibitor medium contradicts the generally accepted concept of a double-stranded structures of the protofibrils as being the only possible ones. Ferry et al. [14] postulated the principle of the “semi-stepwise” combination of fibrin monomer molecules, i.e., “side by side” with a displacement of half of the length. Furthermore, investigations fully confirmed this point of view. The initial act of polymerization is the formation of a dimer giving rise to the lateral contacts between domains D and E (Figure 4). The N-terminal “knobs” of the α-chains in one molecule of a fibrin monomer bind to the a “holes” in the receptor in terminal γC-subdomains of the adjacent molecules. In the trimer, when the C-terminal regions of the fibrin monomers approach one another, a three-domain unit D-E-D is formed leading to appearance of the staggered overlapping rigid rod-like double-stranded fibrin protofibrils. A three-domain unit D-E-D represents the most important structural element of double-stranded protofibrils, because it reflects the peculiarities of the unique multi-domain fibrinogen structure. Despite that the discussed mechanism of fibrin monomer self-assembly is correct, there is some evidence that at the earliest stages the molecules are joined into a dimer in an “end-to-end” fashion [15]. However, this is a brief episode in the general process of polymerization, since the trimers and higher-molecular-mass products are bound in the traditional geometrical fashion. The existence of the equilibrium abnormal single-stranded protofibrils is likely to be explained by an influence of an inhibitor on three-dimensional organization of fibrinogen and fibrin monomer. It is well known that generation of non-covalent bonds – hydrogen, hydrophilic, and electrostatic which participate in the process of self-assembly of fibrin is inhibited in the presence of urea. Moreover, the urea has a destabilizing effect on the protein structure, mainly due to solubilization of the non-polar amino acid residues. It is not a matter of significant structural changes, since the main physicochemical properties of fibrinogen and fibrin monomer coincide, except local conformational transformations in regions of E and D domains. We believe that the strict adjustment of polymerization sites is disrupted. Polymerization site located in the N-terminal region of the α-chains, so-called A “knobs”, of one monomer fibrin molecule is not able to bind to a constitutive complementary-binding site of the a “holes” in terminal γC-subdomain of another adjacent molecule. As a result of this the structural element of double-stranded protofibril, a three-domain unit D-E-D, cannot be formed. In the first turn the inhibitor seems to affect local conformational transformations in the region of terminal γC-subdomain. New reactive sites in D domain are exposed to promote the “end-to-end” self-assembly of fibrin monomer molecules (Figure 4). Damage of the native three-dimension organization of domains D causes an opening of new reaction sites, which are largely different from the polymerization sites a- and “b-holes”. In this respect three-dimensional structure of domain E is more stable. It is known that thrombin binding to fibrinogen is mediated through a fibrinogen recognition site in thrombin [16]. Thrombin binding to the domain E of fibrinogen results in a cleavage and release of the fibrinopeptides from Aα- and Bβ-chains of fibrinogen molecule. The highest possible reliability of fibrinogen conversion into fibrin monomer is needed to guarantee fibrin formation. Therefore it does not seem striking that fibrinogen retains its ability to be hydrolyzed under the action of thrombin at medium conditions far from physiological. When the urea concentration reaches the value of 3.5 M Aα- and Bβ-fibrinopeptides are cleaved from the fibrinogen molecule [17].

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Adapted from Rosenfeld et al., [13].

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Figure 4. Schematic depiction of possible variants of the initial stages of fibrin monomer self-assembly: 1) fibrinogen, 2) fibrin monomer, 3, 4) dimer and trimer formed by lateral joining, 5, 6) dimer and trimer joined “end to end”. αC domains are not shown. Additional explanations are in the text.

From the general thermodynamic considerations single-stranded protofibrils, in which there are only axial contacts, have to be less stable compared with double-stranded protofibrils formed by lateral joining. Upon growing in length single-stranded protofibrils have a tendency to be combined forming more compact double- stranded protofibrils. Singlestranded protofibrils are the rod-like structures, however the mechanism of its structural rigidity remains unclear. Originally, it was thought to have been explained by possible role of the αC domains [12, 13]. The αC domains interact with each other and the central domain E; release of the fibrinopeptides induces intramolecular dissociation of the αC domains and they become interactive, which favors protofibrils association and accelerates elongation of protofibrils and fibrils [6, 7]. The research of mechanism of self-assembly and threedimensional organization of the intermediate soluble forms of X-oligomers in the presence of non-denaturing urea concentration has disproved the possible role of the αC domains in maintenance of structural rigidity of single-stranded protofibrils. X-oligomers were formed from the monomer X- molecules obtained by thrombin action on the X- fragment [18]. The latter is the closest structural homolog of fibrinogen differing by lack of two αC domains [19]. The rod-like structures of single-stranded protofibrils constructed both from the fibrin monomer molecules and from the monomer X- molecules demonstrate that the problem of structural rigidity is unfortunately still far from being understood.

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Mechanism of Aggregation of Fibrinogen Molecules. The Influence of Fibrin-Stabilizing Factor It is known that fibrinogen has open polymerization centers (a and b “holes”) located in its two peripheral D domains. Since complementary centers in E domain containing two polymerization centers (A and B “knobs”) are shielded by fibrin peptides A and B fibrinogen compared with fibrin monomer molecules is unable to take part in the process of spontaneous polymerization. Nevertheless under suitable conditions (for example, low ionic strength, long incubation at 4oC, etc.) fibrinogen can undergo self-association to form structures that are either similar to fibrin [20, 21] or else quasi-globular particles [22]. Fibrin polymers are the preferred substrate for fibrin-stabilizing factor. However, it is shown that fibrinogen is involved in cross-linking reaction in the presence of factor XIIIa [23-25]. This pathway led to complete incorporation of fibrinogen into polymeric matrices. Confocal 3D microscopy showed that these fibrinogen gels were highly disordered structures [2]. On the other hand, fibrils formed in partially cross-linked fibrinogen solutions consisted of two parallel strands, as discerned visually from scanning transmission electron microscopic images and confirmed by mass per unit length fibril measurements [26]. Thus, there are many contradictory and intricate the data regarding the mechanism of generation both unlinked and cross-linked fibrinogen clusters. It is generally accepted that the possibility of covalent fibrinogen crosslinking is due to structural identity of fibrinogen and fibrin molecules. However, fibrinogen molecules could be crosslinked only under the condition of contacts between the peripheral D domains, as in fibrin, but native fibrinogen molecules are incapable of mutual interaction. Hence, the mechanism of enzymatic cross-linking of fibrinogen under the effect of factor XIIIa, which is unambiguously confirmed by biochemical data, remains unclear. On the basis of experimental data on fibrinogen structure and function, it has been proposed that long incubation of fibrinogen molecules in solution under approximately physiological conditions give rise to local conformational rearrangements in region of domain D. This leads to gradual accumulation of so-called “structurally defective” molecules prone to self-association via “end-to-end” interaction, with the eventual formation of flexible fibrinogen homo- or heteropolymers [27-29]. The data from elastic and dynamic light-scattering, and viscosity showed the existence of induction period preceding the aggregation of fibrinogen. However, the duration of this period was shortened in the presence of factor XIIIa. Plots of the angular distribution of lightscattering given in Figure 5 demonstrate a fundamentally different arrangement of the polymer molecules being formed when compared with the rod-like molecules of fibrinogen [27]. By comparing the experimental expression of the average form-factor P(θ) with theoretically derived values for different models represented in Figure 6 [27], a large number of possible structures for homopolymers of fibrinogen can be eliminated from consideration. From the experimental results presented one can see that in the process of aggregation of fibrinogen, single-stranded flexible polymer molecules are formed as we can see from the curves for light-scattering. Starting from the values of average molecular mass of polymer Mω equal to (2,5-3,0)×106 these single-stranded flexible polymer molecules apparently possess a branched structure and tend towards globule formation. Hence, according to the point of view

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being postulated the spontaneous activation and appearance of novel sites situated in COOHregion of domain D occurs upon prolonged storage of fibrinogen in solution, and is responsible for axial contacts (Figure 7). The formation of the dimer is initially brought about as a result of a contact between two structurally modified domains D. The growth of the chain proceeds by joining “defective” fibrinogen molecules. The flexibility of a polymer chain increases in proportion to the number of molecules. On attaining a critical length polymer chain twists into a coil to promote generation more compact structure.

Figure 5. Plots of the angular distribution of light-scattering for unlinked (1-4) and cross-linked (5-7) polymers of fibrinogen at various times: 1) 0 hours, 2) 26 hours, 3) 27 hours, 4) 27,5 hours, 5) 2 hours, 6) 4 hours, 7) 8 hours.

Figure 6. Kratky – Porod plots for rigid thin rods (1), randomly coil-like chains (2), spheres of constant density (3). Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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In the presence of factor XIIIa the structural organization of fibrinogen aggregates do not undergo any major changes. However, there are some characteristic features arising from the formation of ε(γ-glutamyl) lysine bonds catalyzed by the enzyme. The presence of γ-dimers limits structural flexibility of the polymer chains which make polymer chains twist into a coil on attaining a longer critical length. The covalent linkage of α-chains during the later stages of aggregation, as estimated by an increase in the expression 1/P(θ), is related to a decrease in the size of cross-linking polymers compared with the unlinked ones having the same number of connecting links. The hydrodynamic data on the relationship of a diffusion coefficient, intrinsic viscosity and the average molecular mass of polymer confirmed these results. Thus the conclusion is made that the process of aggregation of fibrinogen molecules taking place either in the absence or in the presence of factor XIIIa, involves the formation of flexible branched structures with dense packing of the segments, similar to highly hydrated globular particles impermeable to the solvent.

Adapted from Rosenfeld, Vasil’eva, 1991. Figure 7. Succession of possible stages of assembly of fibrinogen aggregates. 1) native fibrinogen molecule, 2) “defective” fibrinogen molecule, 3) fibrinogen dimer formation, 4) fibrinogen trimer formation, 5) emergence of flexible chain molecules which have a tendency to compaction, 6) generation of γ-γ dimers in the presence of factor XIIIa. αC domains and α-polymers are not shown. Additional explanations are given in the text.

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Factor XIIIa accelerates the formation of fibrinogen aggregates. Based on the above, one possible explanation is that there is a stabilization of intermediate easily dissociated protein complexes owing to the formation of the covalent ε(γ-glutamyl) lysine bonds. The intriguing feature of the γ-chain links in fibrinogen not shared by fibrin should be noted. The data of electrophoresis show that at long incubation of fibrinogen in the presence of factor XIIIa a certain proportion of the γ-chains has still to be intact (Figure 8). By enzyme stabilization of fibrin it is known that the γ-chains of the monomers are almost completely involved in crosslinking [2]. The formation of double-stranded profibrils must therefore be crucially important, monomer molecules being rigidly attached and exactly oriented to one another in a unique way such that the carboxyl-terminal regions of the γ-chains had the chance to complement each other and ensure close contacts between them. In contrast to this the fibrinogen clusters, as it was shown, form a set of coiled, tangled chains with a variety of conformations. The fibrinogen molecules interacting “end-to-end” construct linear polymers that retain the ability to rotate about the covalent bonds. The attraction of the links fixed a series of “defective” conformations, following only a certain proportion of the γ-chains are susceptible to the enzyme cross-linking. The process of fibrinogen aggregation in a solution is completed by formation of particles differing in their structure from the rod-like protofibrils of fibrin. Factor XIIIa proved to accelerate to a considerable degree the formation of the fibrinogen highmolecular-mass assemblies causing apparently irreversible structural transformations in domain D. These local spatial transformations represent an example of the mechanism of the molecular aging.

From Rosenfeld, Vasil’eva, 1991. Figure 8. The electrophoresis data of reduced samples of fibrinogen polymers in the presence of factor XIIIa: 1) 0 hours, 2) 1 hours, 3) 2 hours, 4) 4 hours, 5) 8 hours. (αp-polymers of α-chains, γ-γ, dimers of γ-chains).

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Structural Modification of Fibrinogen as a Result of Free-Radical Oxidation The physicochemical mechanism of molecular aging of fibrinogen (i.e. accumulation of its “defective” molecules) needs to be clarified. Among possible factors responsible for the formation of these structurally “defective” molecules, the involvement of fibrinogen in the spontaneous free radical oxidation appears to be the most relevant. Indeed many amino acid residues of proteins are sensitive to oxidation by various reactive oxygen species (ROS). Free radical oxidation of proteins can be accompanied by cleavage of the polypeptide chains, modifications of the amino acid residues, and protein conversion into the derivatives highly sensitive to a proteolytic degradation [30, 31]. Proteins subjected to the oxidative modification may be accumulated in the body as result of oxidative stress and various diseases [32]. It was shown that fibrinogen is at least 20 times more sensitive to the oxidative modification than other major plasma proteins (albumin, immunoglobulins, transferrin, and ceruloplasmin) [33]. Fibrinogen is easily involved in the reactions of free radical oxidation; this results in the formation of the oxidized forms of this protein, which differ from the native form by both chemical composition and structural organization. This causes changes in the functional properties of fibrinogen. During a free radical attack it can form a macromolecular cluster due to formation of noncovalent bonds [34]. Oxidized fibrinogen inhibits the formation of a fibrin clot catalyzed by thrombin [35]. One may suggest that the abovementioned properties of the oxidized fibrinogen are determined by conformational conversions in its molecules accompanied by the appearance of the new reaction centers. We do believe that some of these reaction centers are the self-assembly sites, which open during incubation of native fibrinogen in a solution. In other words the physicochemical mechanism of the molecular aging of fibrinogen is determined by processes that are identical to either its induced (oxidation) or spontaneous oxidation (during incubation of this protein in a solution). Fibrinogen has undergone a free-radical oxidation induced by the action of ozone [36]. The data of electrophoresis of the reduced samples of fibrinogen oxidized by ozone show that all three polypeptide chains of fibrinogen retain the same molecular masses as compared to native fibrinogen (Figure 9). In the presence of the factor XIIIa, fibrinogen polypeptide chains become subject to the covalent cross-linking, which is manifested as (in) the formation of γdimers with a molecular mass of 95 kDa and Aα-polymers with a molecular mass over 500 kDa, readily identifiable by electrophoresis. The involvement of γ- and Aα-polypeptide chains of ozonized fibrinogen into cross-linking is accelerated, compared to the control, with γdimers and Aα-polymer accumulating at higher rates. Since Bβ chains do not participate in the covalent cross-linking their content remains unchanged. The data of electrophoresis suggest that oxidized fibrinogen is a more preferable substrate for the factor XIIIa, compared to a native fibrinogen. The IR-spectroscopy data indicate that increasing fibrinogen oxidation during ozone treatment leads to a proportional increase in the number of hydroxyl groups (3550–3450 cm– 1 ) and also carbonyl and carboxyl groups (1740–1700 cm–1) (Figure 10). This fact suggests that the oxidant penetrates into amino acid residues, primarily cyclic with C=C double bonds and aromatic, without breaking the ketoimide (amide) bond. The appearance of the absorption bands in 1700 cm–1 and 3230–3450 cm–1 regions is due to the formation of a new amide group resulting in the interaction between ozone and tryptophan or histidine residues with the

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opening of their cyclic fragments. Moreover, changes in the spectrum of the ozonized protein in the region of amide absorption bands indicate that fibrinogen retains its secondary structure; on (in) the other hand, we consider that oxidation leads only to local conformational changes accounted for by the appearance of the new polar groups capable of forming the additional intramolecular hydrogen bonds. The pattern of the chemical modification of fibrinogen is in agreement with the data on the effect of ozone on the amino acid residues of the glutamine synthetase and bovine serum albumin [37].

From Rosenfeld et al., [36]. Figure 9. Electrophoregrams of reduced samples of the intact and ozonized fibrinogen enzymatically crosslinked for (a) 0 min, (b) 30 min, and (c) 120 min: (1, 4, 7) intact fibrinogen; (2,5,8) fibrinogen ozonized by 2×10–6 M of the oxidant; and (3, 6, 9) fibrinogen ozonized by 7×10–6 M of the oxidant.

The data on the formation of the covalently cross-linked fibrinogen polymers and the influence of the oxidation on this process can be interpreted in terms of conformational mobility of the fibrinogen molecule, which is related (concerns) primarily to its peripheral D domains. As noted above native protein molecules cannot interact with each another, whereas oxidation and subsequent local structural rearrangements within the D domain regions make possible their self-association in an “end-to-end” fashion. Optical and hydrodynamic data on the spatial patterns of the fibrinogen polymers appear to support this conclusion. Additional evidence for disturbances in the three-dimensional structure of D domains comes from the fact that the fibrinogen–fibrin conversion is inhibited by free-radical oxidation [35]. Fibrin generated from the “defective” fibrinogen molecules which were formed by either induced

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oxidation [38] or incubation in a solution [39] had a coarser structure characterized by a higher mean mass-length ratio of the polymeric fibers compared to a native fibrinogen This inhibition may result from the retardation of enzymatic removal of fibrinopeptides as well as from disturbances in the polymerization capacity of the monomeric fibrin molecules. However, the rate of fibrinopeptides removal catalyzed by thrombin does not decrease [35], and therefore the conformation of the E domain, which contains thrombin-binding centers apparently remains unchanged. Since self-assembly of fibrin is accounted for by the interaction between D and E domains of its monomers, it is quite probable that an oxidation affects primarily the native D-domain structure, namely γC- or βC- subdomains, which are responsible for binding to the polymerization centers A and B “knobs” located in the domain E. This is also supported by experimental data that an oxidized D-fragment, a fibrinogen degradation product structurally analogous to the D domain enhances the activity of tissue plasminogen activator [40].

Figure 10. Differential IR-spectra of induced-oxidized fibrinogen relative to intact fibrinogen at the different degrees of protein oxidation: 1) 2×10-6 M, 2) 3×10-6 M, 3) 5×10-6 M, 4) 7×10-6 M.

In conclusion, we would like to underline that not all from propositions referring to the role of the conformational mobility of the domain D in the assembly of both fibrinogen clusters and abnormal single-stranded fibrin protofibrils seems to be strictly experimentally substantiated. At present some of them remain assumptions. However, the novel convincing data have been recently obtained regarding to the structural instability of the domain D. Inducted by the ozone free-radical oxidation of D and E fragments of fibrinogen has been studied [41]. UV- and IR-spectroscopy indicated that the fibrinogen fragment D is the most sensitive to free-radical oxidation followed by the local structural changes.

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[9]

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Mosesson, MW. Fibrinogen and fibrin structure and functions. J. Thromb. Haemost. 2005. 3. 1894-1904. Blombäck, B. Fibrinogen and fibrin – proteins with complex roles in hemostasis and thrombosis. Thromb. Res. 1996. 83; 1-75. Madrazo, J, Brown, JH, Litvinovich, S, Dominguez, R, Yakovlev, S, Medved, L, Cohen, C. Crystal structure of the central region of bovine fibrinogen (E5) fragment at 1.4-Å resolution. Proc. Natl. Acad. Sci. USA. 2001. 98. 11967-11972. Medved, L, Litvinovich, SV, Ugarova, TP, Lukinova, NI, Kilikhevich, VN, Ardemasova, ZA. Localization of a fibrin polymerization site complimentary to GlyHis-Arg sequence. FEBS Lett. 1993.320. 239-242. Yang, Z, Mochalkin, I, Doolittle, RF. A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides. Proc. Natl. Acad. Sci. USA. 2000. 97. 14156-14161. Veklich, YI, Gorkun, OV, Medved, IV, Niewenhuizen, W, Weisel, JW. Carboxylterminal portions of the α-chains of fibrinogen and fibrin. J. Biol. Chem. 1993. 268. 13577-13585. Gorkun, OV, Veklich, YI, Medved, IV, Henschen, AH, Weisel, JW. Role of the αC domains of fibrin in clot formation. Biochemistry. 1994. 33. 6986-6997. Muller, MF, Ris, HA, Ferry, JD. Electron microscopy of fine fibrin clots and fine and coarse fibrin films. J. Mol. Biol. 1984. 174. 369-384. Mosesson, MW, Siebenlist, KR, Amrani, DL, DiOrio, JP. Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin. Proc. Natl. Acad. Sci. USA. 1989. 86. 1113-1117. Hewat, EA, Tranqui, L, Wade, RH. Electron microscope structural study of modified fibrin and a related modified fibrinogen aggregated. J. Mol. Biol. 1983. 170. 203-222. McKee, PA, Schwartz, ML, Pizzo, SV, Hill, RL. Cross-linking of fibrin by fibrinstabilizing factor. Ann. NY. Acad. Sci. 1972. 202. 127- 148. Rosenfeld, MA, Gershkovich, KB, Kuznetsov, DV, Meshkov, BB, Gontar, ID. Mechanism of the self-assembly of soluble fibrin oligomers and the role of fibrinopeptides A and B in this process. Mol. Biol. (Russia). 1986. 20. 1098-1110. Rosenfeld, MA, Gershkovich, KB, Kuznetsov, DV. Structural conversions of fibrin oligomers. Mol. Biol. (Russia). 1988. 22. 86-93. Ferry, JD, Katz, S, Tinolo, J. Some aspects of the polymerization of fibrinogen. J. Polymer. Sci. 1954. 12. 509-616. Wiltzius, P, Dietler, I, Känzig, W, Hofmann, V, Straub, PW. Fibrin polymerization studied by static and dynamic light-scattering as a function of fibrinopeptide A release. Biopolymers. 1982. 21. 2205- 2223. Fenton, JW Jr, Olson, TA, Zabinski, MP, Winer, GD. Anion-binding exosite of αthrombin and fibrin(ogen) recognition. Biochemistry. 1988. 27. 7106-7112. Rosenfeld, MA, Kleimenov, AN, Piruzyan, LA. Microcalorimetric investigations of conversion of fibrinogen into fibrin catalyzed by thrombin. Izv. Acad. Nauk. Ser. Biol. 1976. 6. 369-374.

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[18] Rosenfeld, MA, Kostanova, EA, Vasileva, MV, Leonova, VB. The structural conversion of X-oligomers. Izv. Acad. Nauk. Ser. Biol. 1999. 4. 396-402. [19] Weisel, JW, Stauffacher, CV, Bullitt, E, Cohen, C. A Model for Fibrinogen: Domains and Sequence. Science. 1985. 230. 1388-1391. [20] Cohen, G, Stayter, K, Kucera, Hall, C. Polymorphism in fibrinogen aggregates. J. Mol. Biol. 1966. 22. 385-388. [21] Ugarova, TP, Mikhailovskaya, LI, Gornitskaya, OV. Aggregation of molecules of fibrinogen in solution. Ukr. Biokhim. Zhurnal. 1987. 59. 9-14. [22] Becker, CM. Bovine fibrinogen aggregate: electron microscopic observations of quasiglobular structures. Thromb. Res. 1987. 48. 101-110. [23] Kanaide, H, Shainoff, JR. Cross-linking of fibrinogen and fibrin by fibrin-stabilizing factor (factor XIIIa). J. Lab. Clin. Med. 1975. 85. 574-597. [24] Ly, B, Kierulf, P, Jacobsen, E. Stabilization of soluble fibrin/fibrinogen complexes by fibrin-stabilizing factor (FSF). Thromb. Res. 1974. 4. 509-522. [25] Blombäck, B, Procyk, R, Adamson, L, Hessel, B. Factor XIIIa induced gelation of human fibrinogen – An alternative thiol enhanced, thrombin independent pathway. Thromb. Res. 1985. 37. 613-628. [26] Mosesson, MW, Siebenlist, KR, Hainfeld, JF, Wall, JS. The covalent structure of factor XIIIa crosslinked fibrinogen fibrils. J. Struct. Biol. 1995. 115. 88-101. [27] Rosenfeld, MA, Vasil’eva, MV. Mechanism of aggregation of fibrinogen molecules: the influence of fibrin-stabilizing factor. Biomed. Sci. 1991. 2. 155-161. [28] Rosenfeld, MA, Gershkovich, KB. Molecular organization of fibrinogen-fibrin fragment E copolymers. Mol. Biol. (Russia) 1988. 22. 923-933. [29] Rosenfeld, MA, Kostanova, EA, Vasil’eva, MV, Leonova, VB. The mechanism of cross-linking of fibrinogen and its early structural homolog – X fragment. Izv. Acad. Nauk. Ser. Biol. 2001. 3. 293-298. [30] Lushchak, VI. Free-radical protein oxidation and its relationship with the functional state of the body. Biokhimiya. 2007. 72. 995-1017. [31] Stadman, ER, Levine, RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids. 2003. 25, 207-218. [32] Stadtman, ER. Protein oxidation and aging. Free Rad. Res. 2006. 40. 1250-1258. [33] Shacter, E, Williams, JA, Lim, M. Differential susceptibility of plasma proteins to oxidative modification: examination by Western blot immunoassay. Free Rad. Biol. Med. 1994. 17. 429-436. [34] Dijkgraaf, LC, Zaardeneta, G, Corddewener, FW, Liems, RS, Schmitz, JP, de Bont, LG, Milan, SB. Cross-linking of fibrinogen and fibronectin by free radicals:A possible initial step in adhesion formation in osteoarthritis of the femporomandibular joint. J. Oral Maxillofac. Surg. 2003. 61, 101-111. [35] Shacter, E, Williams, JA, Levine, RF. Oxidative modification of fibrinogen inhibits thrombin-catalyzed clot formation. Free Rad. Biol. Med. 1995. 18. 815-831. [36] Rosenfeld, MA, Leonova, VB, Razumovskii, SD, Konstantinova, ML. Mechanism of enzymatic cross-linking of fibrinogen molecules. Izv. Acad. Nauk. Ser. Biol. 2008. 6. 671-679. [37] Berlett, BS, Levine, RL, Stadtman, ER. Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin. J. Biol. Chem. 1996. 271. 4177-4182.

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[38] Rosenfeld, MA, Leonova, VB, Razumovskii, SD, Konstantinova, ML. Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation. Biokhimiya. 2009. 74. 54-61. [39] Rosenfeld, MA, Leonova, VB, Biryukova, MI. The effect of “aging” of fibrinogen molecule on the structure and properties of fibrin. Izv. Acad. Nauk. Ser. Biol. 2007. 4. 394-400. [40] Stief, TW, Marx, R, Heimburger, N. Oxidized fibrin(ogen) derivatives enhance the activity of tissue type plasminogen activator. Thromb. Res. 1989. 56. 221-228. [41] Rosenfeld, MA, Leonova, VB, Shegolihin, AN, Razumovskii, SD, Konstantinova, ML, Bychkova, AV, Kovarski, AL. Oxidized modification of fragments D and E from fibrinogen. Dokl. Akad. Nauk. (in press).

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Chapter 7

Two Faced Members of the Family: The Synucleins Andrei Surguchov VA Medical Center Kansas City, Kansas City, MO, USA and Kansas University Medical Center, KS, USA

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Abstract α, - β- and γ-Synucleins are highly homologous small proteins implicated in neurodegenerative diseases and some forms of cancer. These proteins attracted the attention of many investigators, because of their role in human pathology. Certain soluble α-synuclein oligomers share a common structure with oligomers of other amyloidogenic proteins and peptides, for example, beta-amyloid (Aβ) and prion protein, implying a common mechanism of pathogenesis for several illnesses. γ-Synuclein can also form toxic protein inclusions, although the mechanism of its pathological action is not investigated in the same detail as α-synuclein. β-Synuclein prevents aggregation of αsynuclein and possesses neuroprotective effect. Here we give a brief overview of the structural features of synucleins which may explain why two members of the family (αand γ-synucleins) can be easily converted into oligomeric toxic species, whereas βsynuclein has cytoprotective properties. Among important factors which cause transition of synucleins into pathological molecules are mutations, increased gene dosage (for αsynuclein), post-translational modifications (PTM) and binding to other proteins (for γsynuclein). Oxidative conditions cause the formation of prefibrillar annular γ-synuclein oligomers which can induce aggregation of α-synuclein thus manifesting the property of antichaperone. Substances specifically preventing deleterious protein-protein interactions can be tested as potential drugs for the treatment of neurodegenerative diseases.

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Introduction

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The synuclein family comprises three members:α, β and γ-synuclein which are in the focus of attention of many investigators since the discovery of their role in human diseases. α-Synuclein is implicated in Parkinson disease (PD) and several other neurodegenerative diseases, γ-synuclein is involved in neurodegeneration and cancer, whereas β-synuclein is more known for its beneficial properties preventing the deleterious action of α-synuclein (Bisaglia et al., 2009; Galvin et al., 1999; Hashimoto et al., 2001; Iwatsubo, 2007; Lee et al., 2004; Ninkina et al., 2009). All members of the synuclein family share a characteristically conserved N-terminal domain that consists of 5–6 repeats of the KTKEGV consensus amino acid sequence in the first 87 residues (Figure 1). Synucleins are intrinsically unfolded proteins that can adopt a partially helical structure when they interact with different lipids or bind to membranes [reviewed in Surguchov, 2008]. An intriguing question is why these three homologous proteins with high similarity in amino acid sequence have such different properties? Another exciting question is what are those factors that convert them into toxic substances? For many years since the discovery of the role of α-synuclein in PD and γ-synuclein in cancer the information about these proteins was imbalanced. The majority of studies were focused on revealing the role of these proteins in pathology and mechanisms of their involvement in diseases. On the other hand, their physiological functions remained obscure and their beneficial properties were not investigated in detail and sometimes were ignored.

Figure 1. Schematic drawing of α-, β-, and γ-synucleins structure. Six imperfect repeats (KTKEGV) are distributed throughout most of the amino-terminal half of synucleins. Hydrophobic core (amino acids 71-82) is present in α-and γ-synuclein, but absent in β-synuclein. “Tyrosine signature” consisting of three Tyr residues is present in C-terminal fragment of α-and β-synuclein, but absent in γ-synuclein. Tyr39 plays a critical role in α-synuclein fibrillation [Lamberto et al., 2009]. Methionine residues are important for longrange interaction and secondary structure of α-synuclein. Oxidation of methionine residues in α-synuclein affects electrostatic and hydrophobic interactions and the propensity to fibrillation [Zhou et al., 2009]. Localization of Met38 in a proximal position to Tyr39 in γ-synuclein allows intramolecular oxygen transfer and enhances aggregational propensity.

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We present here a short outline of physiological properties of synucleins and consider some mechanisms of their conversion into pathological species. We discuss post-translational modifications (PTM), binding to other proteins and ligands as factors dramatically changing synuclein properties. Since the majority of studies on synucleins are devoted to α-synuclein pathology in different diseases and are summarized in several recent reviews, we do not discuss these data here (Bisaglia et al., 2009; Iwatsubo, 2007; Surguchov, 2008). We rather propose a new role of the γ-synuclein in neurodegenerative diseases, which may aggregate and form deposits in neuronal and glial cells leading to pathology. In addition, oxidized γsynuclein forms annular oligomers which may fulfill a role of triggers for α-synuclein aggregation and thus may play an important role initiating the cascade of protein deposition.

Physiological Functions of Synucleins

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Despite being associated with several fatal human neurodegenerative diseases, little is known about the normal functions of synucleins. Synucleins are predominantly neuronal proteins which may play a role in the regulation of vesicular turnover, synaptic functions being involved in intracellular processes associated with survival of neurons and their responses to stress. However, two groups of evidence suggest that these functions may be secondary and nonessential and can contribute to the long-term regulation and/or maintenance of presynaptic function: 1) Synucleins are expressed only in vertebrates, while worms, flies and other invertebrate animals lack a recognizable synuclein homologue. 2) Knockout of α-synuclein or even two members of the synuclein family does not cause dramatic consequences in animal models. Mice lacking α-synuclein are phenotypically normal, and only close investigation reveals certain deficits such as a reduction in dopamine (DA) levels, altered synaptic responses and slower replenishment of the docked vesicles by reserve pool vesicles, reduced rearing activity in the open field, etc. [Abeliovich et al., 2000; Cabin et al., 2002; Fleming et al., 2005]. These results suggest that α-synuclein may be required for the genesis, localization, and maintenance of vesicles that make up the reserve or resting pools of presynaptic vesicles. Moreover, double knockouts: α+ β [Chandra et al., 2004] and α+γ synucleins [Papachroni et al., 2005] do not impair basic brain functions or animal survival. Triple knockout caused more significant, but not dramatic effect on mice, affecting synaptic structure and transmission, age-dependent neuronal dysfunction, decreased SNARE-complex assembly as well as diminished survival (Burré et al., 2010; Greten-Harrison et al., 2010). Nevertheless, there are many reports about the effect of synucleins on various cellular functions. The involvement of these proteins in diverse processes may be explained by the peculiar structural organization of synucleins. The lack of a well-defined secondary or tertiary structure of synucleins may play a key role in the molecular recognition of their partners. The major consequences of structural disorder of these proteins are their ability to bind to different partners (proteins, lipids, nucleic acids) with low specificity. As a result of such binding

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synucleins affect a complex network of equilibria in such important cellular processes as signal transduction, cell-cycle interaction, gene expression, protein degradation, vesicular recycling, etc. [Bisaglia et al., 2009; reviewed in Surguchov, 2008].

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α-Synuclein Different experimental approaches show that α-synuclein is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission and presynaptic DA recruitment [Abeliovich et al., 2000; Cabin et al., 2002; Yavich et al., 2004]. In neurons, αsynuclein is associated with presynaptic vesicles and the plasma membrane, possibly via lipid rafts [Fortin et al., 2004]. The modest overexpression of α-synuclein markedly inhibits neurotransmitter release. This inhibition is accompanied by a specific reduction in size of the synaptic vesicle recycling pool, reduced synaptic vesicle density at the active zone, and a defect in the reclustering of synaptic vesicles after endocytosis. Therefore, even moderately elevated levels of α-synuclein produces a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology [Nemani et al., 2010]. These results are in a good agreement with genetic findings according to which duplication and triplication of the α-synuclein gene cause elevation of protein expression and abnormal α-synuclein accumulation leading to Parkinson’s disease [Chartier-Harlin et al., 2004; Ibáñez et al., 2004; Singleton et al., 2003]. One of the normal physiological functions of α-synuclein is associated with its chaperonic activity. For example, α-synuclein acts as a molecular chaperone, assisting in the folding and refolding of synaptic proteins called SNAREs [Chandra et al., 2005]. α-Synuclein cooperates with cysteine-string protein-alpha (CSPalpha) in preventing neurodegeneration. These proteins are crucial for release of neurotransmitters at the neuronal synapse, vesicle recycling, and synaptic integrity. Acting as a chaperone on the presynaptic membrane interface, α-synuclein protects nerve terminals against injury. Another α-synuclein function is related to DA metabolism, including DA synthesis, storage, release, and uptake. The regulatory effect of α-synuclein on DA metabolism may reduce cytoplasmic DA, thereby limiting its conversion to highly reactive oxidative metabolites [Yu et al., 2005]. Although many studies have revealed that α-synuclein is the culprit leading to neuronal cell death, the increasing number of investigations have demonstrated a neuroprotective role of this protein. Under physiological conditions α-synuclein presents a deterrent to the accumulation of other proteins whose deposition might be more toxic to the cell [Kallhoff et al., 2007]. It is very probable that the scenario of α-synuclein behavior, including which properties it will manifest – neurotoxic or neuroprotective, depends on the presence of PTM on its polypeptide chain and the level of its expression. The role of only one PTM – phosphorylation of serine 129 associated with α-synuclein aggregation is extensively investigated [Fujiwara et al., 2002; Iwatsubo, 2007; Kragh et al., 2009; Pronin et al., 2000], while function of site-specific oxidation, nitration, ubiquitination, sumoylation, Oglycosylation in modifying synuclein properties is an emerging area of investigation.

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Therefore, the cells may easily tolerate the reduction of α-synuclein level or even its absence, but are very sensitive to higher levels of expression and to its modifications by mutations or PTM.

β-Synuclein β-Synuclein is a member of the synuclein family that lacks the non-amyloidogenic (NAC) domain responsible for the aggregation of α-synuclein (Figure 1). This protein exhibits the properties of a random coil in solution [Uversky et al., 2002] and can be considered a non-amyloidogenic homolog of α-synuclein. β-Synuclein acts as a physiological inhibitor of α-synuclein aggregation [Hashimoto et al., 2001]. Its neuroprotective effect involves direct interactions between β-synuclein and serine threonine kinase Akt [Hashimoto et al., 2004]. The information about the physiological role of β-synuclein is scarce. Antiapoptotic properties of β-synuclein in neurons may be associated with drastic downregulation of p53 expression and activity. β-Synuclein also restores α-synuclein antiapoptotic function and decreases both caspase 3-like activity and immunoreactivity [da Costa et al., 2003]. In addition, β-synuclein reduces proteasomal inhibition by α-synuclein [Snyder et al., 2005]. Overall, a protective function of β-synuclein can be explained by a combination of low propensity to aggregate, ability to reduce the aggregation of α-synuclein and its effect on proteasomes.

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γ-Synuclein The data about physiological function of γ-synuclein are sketchy. It influences neurofilament network integrity [Buchman et al., 1998] and reduces neurite outgrowth [Surgucheva et al., 2006], however, the mechanism of both effects is elusive. γ-Synuclein upregulates MAPK and Elk-1 [Surguchov et al., 2001], and affects the expression of other genes [Surgucheva et al., 2003] through binding with transcriptional factors [Surgucheva and Surguchov, 2008]. There are some controversial data about γ-synuclein propensity to aggregate and affect the aggregation of other proteins. On the one hand γ-synuclein inhibits α-synuclein fibril formation in in vitro experiments. On the other hand, biophysical studies show that γ-synuclein itself has high propensity to aggregate [Uversky et al., 2002]. These data go in line with the findings of γ-synuclein inclusions in histopathological lesions within neuron bodies, axons and glial cells in several human neurodegenerative disorders, such as neurodegeneration with brain iron accumulation, type 1, Lewy body dementia [Galvin et al., 1999 and 2000], glaucoma [Surgucheva et al., 2002] and in transgenic models [Ninkina et al., 2009; Wang et al., 2004]. Interestingly, inclusions of γ-synuclein in the glial cells of the optic nerve of glaucoma patients look similar to the deposits of α-synuclein in the optic nerve glia in mice overexpressing α-synuclein (Figure 2 A and D). γ-Synuclein is highly expressed in retinal ganglion cells [Surgucheva et al., 2002 and 2008; Soto et al., 2007] and its pathological accumulation and deposition in glial cells of the optic nerve in glaucoma (Figure 2D)

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[Surgucheva et al., 2002] suggests that this ocular disease can also be classified as γsynucleinopathy.

Figure 2. Accumulation and deposition of synuclein in glial cells of the optic nerve. A and B immunohistochemical staining of the optic nerve by α-synuclein antibody from transgenic mouse overexpressing α-synuclein (A) and wild-type mouse (B). C and D - longitudinal sections of the human postlamina part of the optic nerve. γ-Synuclein immunopositive staining of the optic nerve of control individual (C) and patient with glaucoma (D) Scale bars - 50 μm. The arrow-heads in C indicate nerve bundles, the arrows in D show γ-synuclein immunopositive deposits in glial cells.

Transgenic mice overexpressing γ-synuclein develop severe neurological pathology associated with its deposition in cytoplasmic and axonal lesions. Importantly, γ-synuclein positive cytoplasmic inclusions in these mice also looked similar to α-synuclein-positive deposits observed in Lewy body diseases and were Congo Red and Thioflavin S positive. Linear fibrils of γ-synuclein structurally similar to α-synuclein fibrils were also observed [Ninkina et al., 2009].

Interaction of Amyloidogenic Proteins Although it has been known for a long time that protein aggregation underlies a wide range of human disorders, the mechanisms controlling such aggregation are elusive. Protein aggregation is usually initiated by the establishment of anomalous protein-protein interactions through aggregation-prone regions. In some cases, the generation of an aggregation-prone state can be triggered or enhanced by a seeding process. The overlap of clinical and neuropathological features of several neurodegenerative diseases (for example, Alzheimer’s

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disease and Parkinson’s disease) described in the literature [Kurosinski et al., 2002; Lee at al., 2004] raised the question of whether different amyloidogenic peptides may interact with each other. The identification of a 35-amino acid fragment of α-synuclein in the amyloid plaques in DLB brain [Uéda et al., 1993] supports a possibility of Aβ and α-synuclein interaction. The results of immunopathological studies confirmed that Aβ promotes accumulation of both α-synuclein [Masliah et al., 2001] and tau [Obi et al., 2008]. Later it was demonstrated that the formation of amyloid composed of different proteins could affect each other directly or indirectly in some cases contributing to the overlap in clinical and pathological features of neurodegenerative diseases (Giasson et al., 2003). Interestingly, although Aβ exerts a prominent effect on α-synuclein accumulation, α-synuclein does not alter the deposition of Aβ into plaques or the development of plaque-associated neuritic dystrophy [Masliah et al., 2001]. Moreover, in a transgenic mouse model of Alzheimer's disease α-synuclein deficiency caused a significant increase in plaque load in all areas of the forebrain, suggesting that αsynuclein may serve as a chaperone helping the cells to clear protein deposits [Kallhoff et al., 2007]. The existence of Aβ -α-synuclein interactions was demonstrated by multidimensional NMR spectroscopy [Mandal et al., 2006]. Another example of the interaction of different amyloidogenic proteins is a synergistic action of tau and Aβ pathology in Alzheimer’s disease [Götz et al., 2010] and colocalization of tau and α-synuclein in amygdala of Parkinsonism-Dementia Complex Patients of Guam [Forman et al., 2002], familial Alzheimer’s disease [Lippa et al., 1998], neurodegeneration with brain iron accumulation [Saito et al., 2000] and other diseases. A direct interaction between α-synuclein and tau may stimulate the phosphorylation of tau by protein kinase A [Jensen et al., 1999]. Therefore, different amyloidogenic proteins can interact directly by engaging synergistic neurodegenerative pathways. One of such proteins may fulfill a role of a core (or antichaperone) for the aggregation of the other protein(s).

Oxidized γ-synuclein is Toxic and Acts as an Antichaperone As shown in Figure 2 not only α-synuclein, but also γ-synuclein is prone to aggregation and formation of inclusions. Furthermore, α- and γ-synucleins are colocalized in certain cell types [Jeannotte et al., 2009; Ninkina et al., 2003] and heteromeric complexes containing both proteins are described [Wersinger and Sidhu, 2009]. Therefore, we analyzed the aggregation pattern of all three members of the synuclein in vitro. Since synuclein aggregation is slow, we used neurotransmitter dopamine (DA) to enhance the process of aggregation. As a result of such incubation all three forms of synucleins were partially converted into SDS-resistant tetramers (molecular weight ~65 KDa) and high-molecular weight oligomeric forms (molecular weight ~300 KDa)(Figure 3). Previously it was demonstrated that Tyr39 plays an essential role in α-synuclein fibrillation [Lamberto et al., 2009]. In γ-synuclein Tyr39 is located in adjacent position to Met38 (Fig. 1). In proteins with Tyr and Met in neighboring positions an efficient intramolecular oxygen transfer occurs to give dioxygenated derivatives with one oxygen on the Tyr and the other

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forming methionine sulfoxides [Nagy et al., 2009]. In addition, in the structure of micellebound α-synuclein (the PyMOL Molecular Graphics) amino acid residues Leu38 and Tyr39 (corresponding to Met38-Tyr39 in γ-synuclein) are located in a linker connecting α-helices [Surguchov, 2008] between amino acids for which mutations (A30, E46 and A53) are described. Thus, these residues are exposed on the surface of protein and easily susceptible to posttranslational modifications, which may affect the propensity of the protein to aggregation. So we hypothesized that Met38-Tyr39 in γ-synuclein play an important role in physiological properties of γ-synuclein. To check this hypothesis we substituted nucleotides in γ-synuclein cDNA corresponding to these amino acids to convert them to alanine codons. The substitution was carried out using “Site directed mutagenesis kit” (Stratagene). We found that expression of the wt-γ-synuclein significantly slowed down the growth of E.coli cells compared to the growth of cells transformed by an empty vector (Fig. 4). The difference in the growth rate becomes most significant after the addition of IPTG to the medium, which enhances protein expression. Transformation by the cDNA with substitution of Met, Tyr or both amino acids on alanine(s) restored the growth rate. (Fig. 4). The results of these experiments show that Met38 and Tyr39 play a key role determining toxic properties of γ-synuclein, most probably due to their ability to be oxidized because of their neighboring location which allows the intramolecular oxygen exchange. In addition, substitution of these amino acids also prevents the aggregation of γ-synuclein, while oxidized γ-synuclein fulfills a role of a core inducing aggregation of α-synuclein [Surguchov et al., 2011, in preparation].

Figure 3. Aggregation of α-, β-, and γ-synuclein. The results of Coomassie staining of α-synuclein (lanes 2 and 3), β-synuclein (lanes 4 and 5) and γ-synuclein (lanes 6 and 7). The protein samples were incubated at 370 overnight in the presence of DA (lanes 3, 5 and 7) or buffer (lanes 2, 4 and 6). The samples were subjected to the electrophoresis in 12% polyacrylamide gel in the presence of SDSNa and stained by Coomassie R250. Incubation in the presence of DA causes the conversion of monomeric forms of synucleins in tetramers with molecular weight ~65 KDa and aggregates with molecular weight ~300 KDa (arrow).

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These results can be regarded as evidence that Met38 and Tyr39 in proximal position play a key role in determining γ-synuclein aggregation pattern and toxicity.

Figure 4. Effect of the wt- and mutant γ-synuclein on the growth of E. coli. Optical density A600 was measured. IPTG was added after 4 h of cell growth on LB media. 1 – transfection with an empty vector; 2 - Met –Ala substitution; 3 – Tyr- Ala substitution, 4 – Met Tyr-AlaAla substitution. Wt – transfection with a vector containing wt- γ-synuclein.

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Conclusion Here we show that two members of the synuclein family, e.g. α- and γ-synuclein can cooperate in the formation of protein deposits in such a way that oxidized γ-synuclein is a core for α-synuclein aggregation. This may be an important mechanism initiating protein inclusion formation, because these two proteins are colocalized in different brain regions and other tissues. Previously, a possibility that the interaction of amyloidogenic proteins may play a significant role in pathology was discussed for several proteins [Giasson et al., 2003], including α-synuclein and tau [Lee et al., 2004], Aβ and α-synuclein [Uéda et al., 1993]. γSynuclein itself forms deposits in glial cells in the brain [Galvin et al., 1999 and 2000] associated with neurodegeneration with brain iron accumulation, type 1 and in the optic nerve associated with glaucoma [Surgucheva et al., 2002]. A new finding described here is that after oxidation γ–synuclein forms annular oligomers and acquires the property of antichaperone or “sick chaperone”, inducing aggregation of αsynuclein and probably other proteins. The presence of only one tyrosine residue in γsynuclein (compared to four in α-and β-synucleins) and localization of this tyrosine in the adjacent position to methionine (M38Y39) makes this protein especially vulnerable to oxidation and presumably nitration. The oxidative modification of γ-synuclein may take place not necessarily in the presence of DA, since in vivo reactive oxygen species (ROS) are generated by mitochondria, nitric oxide synthase, arachidonic acid metabolism, xanthine oxidase, monoamine oxidase and P450 enzymes, etc. The high metabolic rate of neurons implies a high baseline ROS production.

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An important role of Tyr39 in α-synuclein fibrillation was shown previously [Lamberto et al., 2009]. Methionine residues play an essential role in determining the secondary and tertiary structure of synucleins, because they are involved in electrostatic and hydrophobic long-range interactions of their polypeptide chains and methionine oxidation affects their propensity to fibrillation [Zhou et al., 2010]. The defective chaperones (or chaperone pathology) play an etiological role in several pathology [Macario and Macario, 2007]. Therefore, the diseases in which oxidized γ-synuclein manifests properties of an enhancer of α-synuclein aggregation may be called not only synucleinopathies, but also chaperonopathies. The hypothesis about the dual role of α- and β-synucleins as chaperone and antichaperone and their role in the stimulation of adaptive evolution was put forward earlier [Fujita et al., 2006]. Over the last two decades, there has been an increasing interest and certain success in developing therapeutics that inhibit specific protein-protein interactions. Further progress in this field may bring efficient inhibitors of protein-protein interaction and protein aggregation suitable for the treatment of neurodegenerative diseases.

Acknowledgment This study is supported by the following grants: VA Merit Review, Alzheimer’s Disease Research Center, Reeves Foundation and The Glaucoma Foundation.

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References Abeliovich, A; Schmitz, Y; Fariñas, I; Choi-Lundberg, D; Ho WH; Castillo PE; Shinsky N; Verdugo JM; Armanini M; Ryan A; Hynes M; Phillips H; Sulzer D; Rosenthal A. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron, 2000 Jan; 25 (1): 239-52. Bisaglia, M; Mammi, S and Bubacco L. Structural insights on physiological functions and pathological effects of α-synuclein. The FASEB Journal, 2009, 23, 329-340. Buchman, VL; Adu, J; Pinon, LG; Ninkina, NN and Davies, AM. Persyn, a member of the synuclein family, influences neurofilament network integrity. Nat. Neurosci., 1998, 1, 101–103. Burré, J; Sharma, M; Tsetsenis, T; Buchman, V; Etherton, MR; Südhof, TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science, 2010 Sep 24; 329 (5999): 1663-7. Cabin, DE; Shimazu, K; Murphy, D; Cole, NB; Gottschalk, W; McIlwain, KL; Orrison, B; Chen, A; Ellis, CE; Paylor, R; Lu, B, and Nussbaum, RL. Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. J. Neurosci. 2002, 22, 8797–8807. Chandra, S; Fornai, F; Kwon, HB; Yazdani, U; Atasoy, D; Liu, X; Hammer, RE; Battaglia, G; German, DC; Castillo, PE; Südhof, TC. Double-knockout mice for alpha- and beta-

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Chapter 8

Inclusion Bodies: A New Concept of Biocatalysts Neus Ferrer-Miralles1,2,3, Mónica Martínez-Alonso1,2,3, Antonio Villaverde1,2,3 and Elena García-Fruitós3,1,2*

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1

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain 2 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain 3 Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain

Nowadays, many enzymes are used as catalysts in a wide range of bioprocesses in both chemical and pharmaceutical industries. Although the use of these biocatalysts has an enormous potential, the high cost of catalyst production is the most important bottleneck of the whole process. In this context, in the last decades, many enzyme immobilization strategies have been developed to favour the economical viability of enzymatic reactions in industrial processes. In this format, biocatalysts are then reusable and stable even under harsh conditions and can be tailored for different applications such as bioremediation, as biosensors and in the production of complex compounds, among others [1]. The most common immobilization methods can be divided into two categories depending on whether biocatalyst immobilization is support-dependent or support-independent [2]. Immobilization based on the interaction of the enzyme with a support can be achieved by enzyme entrapment using a polymer network as a support, enzyme encapsulation and enzyme support-based immobilization onto a prefabricated support. Although the main advantage of strategies such as enzyme entrapment or encapsulation is the protection of the catalyst from direct contact with the environment, both methods also have a major limitation regarding mass transfer. Moreover, considering that in support-based immobilization around 90-99% of * Correspondence: [email protected], Phone: +34 935812864; Fax: +34 935812011

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the mass and volume of the catalyst is the support itself, an important drawback is observed concerning enzyme dilution and reduction of specific activity. In addition, the interaction of the enzyme with the solid support results in a significant reduction of overall activity of the immobilized biocatalyst due to unspecific interactions which can ultimately affect the accessibility of the substrate to the active site by steric hindrance, or by inducing a structural change in the active site [3, 4]. In order to improve specific activity of the immobilized enzyme, more rationale protocols have been developed to direct the binding of the biocatalyst to the solid support through the use of linkers that are designed to specifically bind to previously identified regions of the protein. In that case, the directed interactions have a reduced impact on enzyme-mediated catalysis [5]. On the other hand, carrier-free immobilization protocols such as cross-linked enzyme in solution, in crystals or in aggregated forms have been developed [6-8]. The main idea of this methodology is to form highly pure biocatalyst networks by covalent protein-protein interactions produced by homo-bifunctional reagents such as glutaraldehyde in which structural rigidity of the protein is maintained [9]. Therefore, enzyme cross-linking has been developed in many variants including cross-linked enzyme crystals method (CLECs) [6], cross-linked enzyme directly in solution (CLEs) [6] and cross-linked enzyme aggregation (CLEA) [6, 10]. All these methodologies show advantages such as enzyme stabilization and improved resistance. However, cross-linking is still a trial-and-error process (a specific protocol has to be designed for each enzyme), thus becoming a time-consuming and often expensive procedure. In addition, the low diffusion of large molecules between proteins poses a drawback when developing this technology. Furthermore, recent studies show that bacterial inclusion bodies (IBs) can be successfully used as biocatalysts in many processes [11-15], becoming an appealing alternative to the traditionally used immobilization methods mentioned above. Although IBs have been historically described as insoluble protein clusters essentially formed by misfolded and, therefore, inactive proteins [16], over the past few years new evidences have proved the existence of active protein embedded in such aggregates, which clearly contradicts this dogma [11-13, 15, 17-18]. Specifically, recent insights into the structure and physiology of these aggregates reveal that at least a fraction of the polypeptides embedded as IBs exhibit native-like structure [19], which results in enzyme-formed IBs being at least partially active. Additionally, IBs are highly pure aggregates, essentially formed by the overproduced recombinant protein. These structures, which are highly porous and hydrated, are also mechanically stable, cheap and easy to produce [20]. The incorporation of IBs in industrial catalysis, as we previously mentioned, could represent an important conceptual shift in the biotechnological market. In this context, many reports which prove the obvious potential of these proteinaceous aggregates have recently been published. Interestingly, different types of enzymes such as galactosidases [11, 21], reductases [11], oxidases [18], kinases [13, 14], phosphorylases [22] and aldolases [15] have been tested, all of them being able to catalyze specific processes when used in their aggregated form (table 1). The obtained results seem to suggest that substrate can diffuse easily in the core of such particles, probably due to the high porosity of these aggregates [11, 21]. In addition, once the reaction is completed, IBs can easily be removed from the reaction mixture by a simple low speed centrifugation and, moreover, they can be used in more than one reaction cycle (table 1). Furthermore, it should also be noted that only negligible protein

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amounts are released from IBs during reaction processes in aqueous solutions [21], which makes them not only mechanically but also biologically stable structures. Another clear advantage of the use of IBs for industrial biocatalysis becomes apparent when comparing their easy purification process to the long procedures needed for soluble protein versions. For instance, the purification of sialic acid aldolase requires a 7-step procedure (which includes ammonium sulphate precipitation of the crude cell extract followed by heat treatment, ion exchange, gel filtration, hydroxyapatite chromatography and chromatofocusing) to render the protein pure enough to form crystals [23]. A similar method is used for the purification of maltodextrin phosphorylase obtained in recombinant Escherichia coli. In this case, the purification procedure must also deal with the removal of all traces of bacterial phosphatases, in order to avoid cleavage of the reaction product [24]. Thus, these tedious procedures make purification processes difficult and expensive, and for this reason alternative methods have been explored. Whole-cell biocatalysis eliminates the need for purified proteins, and renders more stable enzymes as they are protected by the cell envelope. This approach has allowed the production of sialic acid by coupling bacterial whole cells producing the required enzymes either upon chemical (e.g. IPTG) or thermal induction [25-26]. However, whole-cell biocatalysis may result in deleterious or uncontrolled sideeffects of other cell components on the recombinant product, and for this reason the use of the enzyme as IBs may prove more convenient. In fact, by fusing the cellulose binding domain from Clostridium cellulovorans (which acts as an aggregation domain) at the N-terminus of sialic acid aldolase, functional IBs were produced, and their purity was comparable to that obtained for the soluble version resulting from the long procedure described above [15]. Furthermore, for poorly soluble enzymes, their straightforward use as IBs also represents an advantage. This is the case of polyphosphate kinase, which is only 30% soluble. In addition, this enzyme has also shown to be sensitive to immobilization and, being quite unstable, needed to be refreshed for every new reaction [27], which also supports the use of the IB version for biocatalysis. Moreover, the specific activity of the enzyme embedded in IBs can be influenced by different parameters such as culture conditions (temperature and curve growth phase) and genetic background of the producing strain [20, 28-30]. Besides, the size of these aggregates can also be modulated using different genetic backgrounds and also controlling the formation time of the IBs [20, 28]. For instance, strains lacking the main chaperone DnaK or the Lon or ClpP proteases produce larger IBs than a wild type strain. Therefore, since size and enzymatic activity [20, 28] of IBs can be easily controlled, a fine tuning of the final protein cluster (IB) of the enzyme of interest can be done. Also, by being subjected to conventional immobilization processes enzymes lose part of their activity, which is in fact strongly influenced by the mode of immobilization and carrier properties. Hence, the precipitation of the target enzyme into insoluble particles such as inclusion bodies becomes a perfect choice.

Acknowledgments The authors appreciate the financial support received through grants BIO2007-61194 from MICINN and 2009SGR-108 from AGAUR. We also acknowledge the support of the

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CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. AV was distinguished with an ICREA ACADEMIA award. Table 1. Enzyme-based inclusion bodies used to catalyze bioprocesses Enzyme β-galactosidase Human Dihydrofolate Reductase (hDHFR) Polyphosphate Kinase

Process ONPG and CPRG hydrolysis NADPH to NAPP+ conversion ATP/NTP synthesis

Maltodextrin Phosphorylase

Degradation of soluble starch

Sialic Acid Aldolase

Production of neuraminic acid Conversion of α-amino acid into α−keto acid, H2O2, NH2 Cytidine monophosphate Nacetylneuraminic synthesis

D-amino acid Oxidase Polyphosphate Kinase Cytidilate Kinase

Recyclable nd nd

Reference [11, 21] [11]

17 reaction cycles 10 reaction cycles 20 reaction cycles nd

[13]

[18]

nd

[14]

[22] [15]

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References [1]

[2] [3] [4]

[5] [6] [7] [8]

Fernandez-Lafuente,R. Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst. Journal of Molecular Catalysis B Enzymatic 62, 197-212 (2010). Xie,T. et al. Recent advance in the support and technology used in enzyme immobilization. African Journal of Biotechnology 8, 4724-4733 (2009). Petkar,M., Lali,A., Caimi,P., & Daminati,M. Immobilization of lipases for non-aqueous synthesis. Journal of Molecular Catalysis B-Enzymatic 39, 83-90 (2006). Yang,T.K., Fruekilde,M.B., & Xu,X.B. Applications of immobilized Thermomyces lanuginosa lipase in interesterification. Journal of the American Oil Chemists Society 80, 881-887 (2003). Wong,L.S., Khan,F., & Micklefield,J. Selective Covalent Protein Immobilization: Strategies and Applications. Chemical Reviews 109, 4025-4053 (2009). Roessl,U., Nahalka,J., & Nidetzky,B. Carrier-free immobilized enzymes for biocatalysis. Biotechnol. Lett.(2009). Nahalka,J. & Gemeiner,P. Thermoswitched immobilization - A novel approach in reversible immobilization. Journal of Biotechnology 123, 478-482 (2006). Brady,D. & Jordaan,J. Advances in enzyme immobilisation. Biotechnol Lett. 31, 16391650 (2009).

Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

Inclusion Bodies: A New Concept of Biocatalysts [9] [10] [11] [12] [13]

[14] [15] [16] [17]

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Iyer,P.V. & Ananthanarayan,L. Enzyme stability and stabilization - Aqueous and nonaqueous environment. Process Biochemistry 43, 1019-1032 (2008). Sheldon,R.A. Cross-linked enzyme aggregates (CLEAs): stable and recyclable biocatalysts. Biochem. Soc. Trans. 35, 1583-1587 (2007). Garcia-Fruitos,E. et al. Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microbial Cell Factories 4, (2005). Nahalka,J., Mislovicova,D., & Kavcova,H. Targeting lectin activity into inclusion bodies for the characterisation of glycoproteins. Mol Biosyst. 5, 819-821 (2009). Nahalka,J., Gemeiner,P., Bucko,M., & Wang,P.G. Bioenergy beads: a tool for regeneration of ATP/NTP in biocatalytic synthesis. Artif. Cells Blood Substit. Immobil. Biotechnol. 34, 515-521 (2006). Nahalka,J. & Patoprsty,V. Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3). Org Biomol Chem 7, (2009). Nahalka,J., Vikartovska,A., & Hrabarova,E. A crosslinked inclusion body process for sialic acid synthesis. J Biotechnol 134, 146-153 (2008). Baneyx,F. & Mujacic,M. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol 22, 1399-1408 (2004). Nahalka,J. & Nidetzky,B. Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates. Biotechnol. Bioeng. 97, 454-461 (2007). Nahalka,J., Dib,I., & Nidetzky,B. Encapsulation of Trigonopsis variabilis D-amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme. Biotechnol. Bioeng. 99, 251-260 (2008). Ventura,S. & Villaverde,A. Protein quality in bacterial inclusion bodies. Trends Biotechnol 24, 179-185 (2006). Garcia-Fruitos,E. et al. Surface Cell Growth Engineering Assisted by a Novel Bacterial Nanomaterial. Advanced Materials 21, 4249-4253 (2009). Garcia-Fruitos,E., Aris,A., & Villaverde,A. Localization of functional polypeptides in bacterial inclusion bodies. Applied and Environmental Microbiology 73, 289-294 (2007). Nahalka,J. Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D-glucose1-phosphate. J Ind Microbiol Biotechnol 35, 219-223 (2008). Aisaka,K., Igarashi,A., Yamaguchi,K., & Uwajima,T. Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli. Biochem. J 276 ( Pt 2), 541-546 (1991). Weinhäusel,A., Nidetzky,B., Kysela,C., & Kulbe,K.D. Application of Escherichia coli maltodextrin-phosphorylase for the continuous production of glucose-1-phosphate. Enzyme Microb. Technol. 17, 140-146-146 (1995). Zhang,Y. et al. An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system. Appl. Microbiol Biotechnol 86, 481-489 (2010). Xu,P. et al. Efficient Whole-Cell Biocatalytic Synthesis of N-Acetyl-D-neuraminic Acid. Advanced Synthesis & Catalysis 349, 1614-1618 (2007).

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[27] Liu,Z., Zhang,J., Chen,X., & Wang,P.G. Combined biosynthetic pathway for de novo production of UDP-galactose: catalysis with multiple enzymes immobilized on agarose beads. Chembiochem. 3, 348-355 (2002). [28] Garcia-Fruitos,E. et al. Divergent genetic control of protein solubility and conformational quality in Escherichia coli. J Mol Biol 374, 195-205 (2007). [29] Gonzalez-Montalban,N., Garcia-Fruitos,E., Ventura,S., Aris,A., & Villaverde,A. The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells. Microb. Cell Fact. 5, 26 (2006). [30] Gonzalez-Montalban,N., Natalello,A., Garcia-Fruitos,E., Villaverde,A., & Doglia,S.M. In situ protein folding and activation in bacterial inclusion bodies. Biotechnol Bioeng 100, 797-802 (2008).

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In: Protein Aggregation Editor: Douglas A. Stein, pp. 199-222

ISBN: 978-1-61761-815-4 © 2011 Nova Science Publishers, Inc.

Chapter 9

Comparative Study of Bovine and Ovine Caseinate Aggregation Processes: Calcium-Induced Aggregation and Acid Aggregation María Eugenia Hidalgo1, Manuel A. Mancilla Canales1, Cássia R. Nespolo2, Anselmo D. Reggiardo1, Estela M. Alvarez1, Jorge R. Wagner3 and Patricia Risso*1 Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

1

Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina 2 Departamento de Zootecnia - Centro Educacional do Oeste – Universidade do Estado de Santa Catarina, Chapecó, Brasil 3 Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina

Abstract The solubility of colloidal particles of bovine and ovine caseinate in the presence of calcium was studied by analyzing the colloidal particle size and the protein composition of casein colloidal aggregates remaining in suspension. A comparison between the behaviour observed for bovine and ovine caseinate was carried out. A two-step saltingout process, due to progressive Ca2+ binding to at least different two kinds of sites was observed for both caseinates. The precipitation curves were fitted and the affinity constants and binding site numbers were estimated with an equation based on the concept of Wyman’s linked functions. Ovine caseinate colloidal aggregates formed in the presence of calcium turned out to be less stable and quite bigger than the bovine ones. The binding of calcium to protein residues with some particular characteristics modifies *

Corresponding author: Tel: 54-0341-4804592/97 (int. 253), FAX: 54-0341-4372704, E-mail: phrisso@ yahoo. com.ar

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María Eugenia Hidalgo, Manuel A. Mancilla Canales, Cássia R. Nespolo et al. not only the composition but also the conformational state of caseinates. An aggregation process at low caseinate concentration and an acid-induced gelation process at high protein concentration triggered by the hydrolysis of glucono-δ-lactone were also studied. The effects that variables such as temperature, protein concentration and GDL amount exerted on these processes were analyzed using spectroscopic-based methods and measuring the rheological properties of systems. The time required to initiate particles aggregation decreased in parallel with an increment of temperature, amount of GDL added and caseinate concentration. An increase in caseinate concentration or a reduction of temperature produced gels with a substantial rise in the storage modulus. Modifications of GDL/caseinate ratio did not resulted in significant changes in the rheological parameters determined. The kinetic of the aggregation and compactness degree of ovine caseinate aggregates formed at the end of the acidification process were different from those of bovine source. Acid-induced aggregation and gelation processes were also investigated in the presence of calcium concentrations where no precipitation occurs. The addition of calcium affects the kinetic of both processes and the final state of the aggregates or gels obtained. Consequently, the degree of compactness and average size of the aggregates and rheological properties of gels produced at the end of the acidification process depend on the calcium concentration added.

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Introduction Caseins represent the main protein (76-83%) fraction in bovine and ovine milk. These proteins and their derived salts, the caseinates, are extensively used in food industry because of their physicochemical, nutritional and functional properties that make them valuable ingredients in complex food preparations. Caseins (CN) occur in milk as stable colloidal aggregates known as casein micelles, mainly composed by αS1-, αS2-, β- and κ-CN) [1, 2]. Regarding aminoacidic composition, there is no difference in the amount of residues between the αS1-CN fraction of ovine and bovine caseins whereas αS2 and κ-CN possess a higher number of residues in the case of the casein from ovine source. On the other hand ovine β-CN contains a less amount of amino acid residues than the bovine one [3]. It should highlighted that the number and position in the polypeptide chain of phosphoserine and phosphothreonine residues in CN from both species are similar, except for ovine αS1-, αS2and κ-CN that contain one more phosphoserine residue as part of their primary sequence [2, 4, 5]. Caseinates (CAS) are commonly employed as additives in a great variety of food products because of their high emulsifying, water-binding and gelation capabilities, their heat stability and their contribution to the food texture and juiciness. Therefore, these properties make caseinates useful and desirable ingredients in the preparation of bakery and confectionery products, where they can be used as milk substitutes [6]. Moreover, from a nutritional point of view, caseins are a valued and easily digestible source of all the essential aminoacids and play an important role in the transport of calcium and phosphate; for this reason contributing to a carefully balanced diet [7]. Sheep milk can be used for different purposes and has become a very important resource for the emerging economy in developing geographic areas where it constitutes a major source of special high-quality products [8, 9]. Production of typical dairy products from ewes can provide a profitable alternative to cow milk products owing to their specific taste, texture and

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their natural and healthy properties. Besides, cheeses manufactured from ewes milk are considered gourmet products due to their high added value with a consequent impact on the economy of the productive sector [10]. Among the different types of CN, there are some important features that differentiate them, based on their charge distribution and their propensity to precipitation by the action of Ca2+. κ-CN fraction, insensitive to Ca2+, acts as shell-like structure in the colloidal particles that aims to prevent the other CN from precipitating when Ca2+ is present [11]. Ca2+ binding is a reversible process and has an important nutritional impact as it strongly influences the bioavailability of Ca2+ delivered by these proteins [12]. Changes on the ratio Ca2+:CAS could affect not only the functional properties but also some physicochemical ones such as viscosity, solubility and stability. Hence, it is also reasonable to study how the conformational state of CN and Cas is modified as it is one of the most critical factors that will determine how the functional property is affected [13]. Nowadays consumers have increased the demand of healthy and high nutritional-valued food products. As a result, food industry has undergone an increment in the production of supplemented foods that contain dietary additives such as minerals. However, while it represents a significant benefit from a nutritional perspective, the addition of minerals (e.g. Ca2+) constitutes an inconvenience in the formulation of dairy products because of the reduction on the mineral and protein concentration due to the precipitation caused by the technological treatments in the presence of these mineral species. This unfavourable effect could be, in some extent, reverted by incorporating milk proteins which have the ability to bind Ca2+ and reducing the amount of soluble Ca2+ up to levels that would not alter the stability of the derived product. The stability of milk proteins and the availability of Ca2+ are dependent on the mineral equilibria between caseins and the ions in the soluble phase. Therefore, to analyze the different equilibria involved in the stability of the protein-cosolutes systems is of capital importance considering that the succesful enrichment of food preparations aimed at consumers will depend on the interactions between these two components. Although solubility and colloidal stability of bovine caseins in the presence of calcium ions have been extensively analyzed by different authors from both thermodynamic and kinetic approachs [14, 15], research regarding ovine caseins has not been a full subject for study yet. A dissociation and further aggregation steps of CN fractions due to CAS acidification results in the formation of a gel structure. Casein gels are responsible for most of the rheological/textural properties (i.e. stretch, fracture) of cheese and other dairy products. Rheological properties are monitored as a quality control method in food industry to perform research on the structure/texture of food products [3, 16]. At present, a process that has gained the attention of food industry is direct acidification by the addition of a lactone, such as glucono-δ-lactone (GDL) which allows to overcome some of the difficulties associated with the traditional process of using starter bacteria. In fact, the final pH of the system is has a direct relationship with the amount of GDL added whereas starter bacteria produce acid until they inhibit their own growth as pH becomes lower [17, 18]. Even though rheological characteristics and functional properties of cow milk and its dairy products have been fairly well studied, the information available regarding those of sheep milk products is still scarce [3].

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The aim of this chapter was to describe a comparative study of several functional properties of ovine and bovine caseinates.

Colloidal Stability Test A fixed volume of desired concentration of calcium chloride was added to a protein solution at pH 6.8. The mixtures were maintained in a water bath for 1 hour. Subsequently, these colloidal systems were centrifuged at 1,500 times gravity for 20 min. Precipitates (insoluble casein aggregates) and supernatant (casein colloidal aggregates, CCA) were obtained as individual fractions. CAS precipitation by Ca2+ was interpreted by Farrell et al. [14, 19], using the concept of Wyman’s linked functions [20], assuming that cation binding to the protein is followed by a precipitation step or salting-out process of the lesser soluble calcium caseinate particles formed. This multiple equilibrium is represented as follows:

Kn K' n' 1 p + nCa ← ⎯⎯ → pCan + n' Ca ← ⎯ 1⎯ → p Can Can'

(1)

where p is the unbound protein, n and n’ are the number of Ca2+ moles bound to the specie pCan and pCanCan’ respectively. K n and K' n' are the precipitation or salting out equilibrium 1

1

constants. The mathematical relationship representing the above stoichometry will be:

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Sapp = S0 f ( p) + S1 f ( pCan ) + S1' f (pCan Can' )

(2)

where Sapp is the apparent protein solubility at a given Ca2+ concentration, f(i) are the protein fractional components of species i, S0 the initial concentration of soluble caseinate, S1 is the apparent solubility of pCan and S' is the apparent solubility of pCanCan’. 1

Alvarez et al.,24 developed the following equation, which is used to fit the experimental data of solubility as a function of concentration of Ca2+ as it was found that a second saltingout step takes place as calcium concentration increases due to the presence of a second group of salting-out sites n’) [21]:

Sapp =

S0

1+ K1n [ Ca 2+ ]

n

+

S1 K1n [ Ca 2+ ]

n

1+ K1n [ Ca 2+ ]

n

(S +

' 1

− S1 ) ( K1' ) [ Ca 2+ ] n'

1+ K1' [ Ca 2+ ]

n'

n'

(3)

where K1 and K1' correspond to the first and second salting-out process respectively. CAS solubility data were subjected to non-linear regression using the Levenberg-Marquard algorithm. The CCA that remained in the supernatant fraction after the colloidal stability test were analyzed on their qualitative composition by Urea-SDS-PAGE using a vertical gel system, according to the method of Laemmli [22]. Protein concentration was determined by the

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203

Kuaye method which is based on the ability of strong alkaline solutions to shift the spectrum of the amino acid tyrosine to higher wavelength values in the UV region [23]. The protein concentration remaining in the supernatant (i.e., in colloidal suspension), when precipitation of CAS was induced by increasing total calcium concentration (TCC), and centrifuged at 1,500 times gravity is shown in Figure. 1. The solubility profiles obtained for both CAS showed certain similarities. In fact, they exhibit the presence of a two well defined steps of salting-out. For ovine CAS, precipitation started at low TCC (~3 mmol L-1) and continued until an important protein fraction was precipitated (50 % of total protein at 20 mmol L-1 of TCC) (Figure 1A). Comparing these results to those obtained for bovine CAS (Figure 1B), it can be observed that for the latter precipitation starts approximately under conditions where TCC has increased twofold. In addition, only 35 % of bovine CAS was precipitated at 20 mmol L-1 of TCC. Table I shows the calculated values for the affinity constants (K1, K '1 ) and estimated binding site numbers (n, n′ ) which were determined with a model based on the concept of Wyman’s linked functions by using the Gauss-Newton algorithm from the curves fitted using Eq. (3). In both regression analyses the non-linear model was statistical significative. Besides, a normal error distribution assumption was verified by the Shapiro-Wilks test in both cases.

A

3

2

1 0

5

10

15

20

25

30

-1

TCC (mmol L ) 5

B

Sapp (g L-1)

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Sapp (g L-1)

4

4

3

2

0

5

10

15

20

25

30

-1

TCC (mmol L ) Figure 1. Mean experimental values (n=3) of remnant CAS concentration (Sapp) in the supernatants of mixtures of 5 g L-1 ovine (A) or bovine (B) sodium CAS as a function of the total calcium concentration (TCC), pH 6.8, temperature 25 ºC. The gray lines represent the fitting from Equation 3. Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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Across the first salting-out stage these parameters (affinity constants, number of binding sites) exhibited higher values in the case of ovine caseinate. On the other hand, two different kinds of Ca2+ binding sites can be estimated when the precipitation plots are analyzed. Apparent average binding constants differed in one order of magnitude with the stronger ones (K1) that correspond to the initial step of the precipitation process. Therefore, since it is wellknown that the affinity of phosphoserine residues for Ca2+ is higher than any other binding sites [14, 21], we assume that this first step involves most of the casein phosphoserine residues as higher K1 values provide a clear evidence. However, we cannot disregard the participation of other anionic residues, such as carboxylate groups. Table 1. Average (n=3) values for affinity constants and binding sites when calcium is added to sodium caseinates. Eq. (3) was used for determination. Protein concentration 5 g L-1, 25 °C and pH 6.8 Parameters

K1 (L mol-1)

n

S1 (g L-1)

K1' (L mol-1)

n'

S’1 (g L-1)

Ovine CAS Bovine CAS

300 ± 22 72 ± 2

18 4

3.6 ± 0.1 2.4 ± 0.1

68 ± 2 43 ± 1

4 17

1.6 ± 0.1 1.7 ± 0.1

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As regards the second salting out step (Table I) the average values for both affinity constants were fairly lower, consequently, suggesting the participation of weaker affinity sites. In any case, K1 and K1' values were higher for ovine caseinate compared to the bovine counterpart. A qualitative and quantitative analysis of SDS-PAGE showed for ovine caseinate under all TCC, a prevalent content of α- and β-CN fractions within CCA compared to the amount of κ-CN in the case of (Figure 2A). On the other hand, a lesser ovine κ-CN fraction is observed as compared with the bovine. As regards bovine CAS, a significant α-CN protein precipitation ocurred as it is demonstrated by the small electrophoretic band, and an almost invariable composition for κCN leading, consequently, to an increase of the fractional percentage of β-CN (Figure 2B). Presumably, the representative decrease in α-CN content could be associated to the moment when a “shoulder-like” region appears in the solubility curve. In the second salting-out stage, it can be observed a reduction of α-CN percentage and a corresponding increase in the β- and κ-CN percentage as compared with the absence of Ca2+. We could infer that, even though all the CN precipitate, at last, the α-CN seems to be more susceptible. Similar results were also reported by Pitkowski et al. [24]. On the contrary, this change of composition was not detected in the ovine system probably due to the difference in αS-CN phosphorylated residues between both species. According to the results reported above, at a given temperature and pH, ovine calcium CCA seems to be less stable than the colloidal particles of bovine source. The differences observed might be related to the composition and different proportion of caseins in cow and sheep milks. Sheep milk contains a higher amount of α- and β-CN that in turn have a higher amount of phosphoserine residues capable of binding to Ca2+ [2], a feature that makes ovine caseins more sensitive to lose their stability. Moreover, ovine casein is characterized by a smaller content of κ-CN fraction which is known to act in the stabilization of casein colloidal

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aggregates as mentioned before. Therefore, this diminished stability exhibited by the ovine particles could also be attributed to a lower amount of κ-CN. 1.0

casein fraction

0.8

1.0 0.8

0.6

0.6

0.4

0.4

0.2

Sapp fraction

A

0.2

0.0

0.0 -5

0

5

10

15

20

25

30

-1

TCC (m mol.L ) 1.0

B

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1.0 0.8

0.6 0.4

0.6

0.2

0.4

0.0

Sapp fraction

casein fraction

0.8

0.2 -5

0

5

10

15

20

25

30

-1

TCC (m mol L ) Figure 2. Protein composition (bars) and protein solubility of CCA (●) versus TCC for ovine (A) and bovine (B) caseinate at 5 g L-1. α-CN (■), β-CN (■) and κ-CN (■).

Size Variations of the CCA Particles size changes and/or degree of compactness were followed by the dependence of turbidity (τ) on the wavelength (λ). τ was measured as absorbance (A) in the (400 to 600) nm range, where there is no protein chromophores groups absorbance. For monodispersed particles system of molecular weight M, concentration c, and with a refractive index close to that of the solvent, the turbidity is given by:

⎛ ∂n ⎞ 32 π n ⎜ 1 ⎟ ⎝ ∂c ⎠ where H = 3N λ4 2

τ = HcMQ

2

2 0

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where n0 and n1 are the refractive indexes of the pure solvent and the solution, respectively, N is the Avogadro’s number, and dn1/dc is the specific refractive index increment. The dissipation factor Q results from the internal interference of light scattered by the particle at all the angles θ. The function Q depends on the particle size and can be defined as: π

3 Q = ∫ P(q, R )(1 + cos2θ ) senθ dθ 8 0

(5)

where θ is the dispersion angle of light and P(q,R) is a size factor function of the wavelength vector q and the radio R of the particle [25]. Simulation studies using different models for particle aggregation have highlighted the fractal nature of the colloidal aggregates obtained [26]. For an object with fractal structure, Q can be defined according to the following expression: π

3 Q = ∫ P(q, R) S(q) (1 + cos2θ ) senθ dθ 8 0

(6)

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where S(q) is a structure factor that describes the special arrangement of the dispersion elements or monomers within the aggregate [27]. From Eq. 6, we can obtain, at a constant c, the derived equation:

⎛ ∂ n⎞ ∂ log⎜ n ⎟ ⎝ ∂c⎠ ∂ log τ ∂ log Q = +2 −4= β+γ −4 ∂ log λ ∂ log λ ∂ log λ

(7)

The parameter β, as a consequence, can provide useful information related to the size and degree of compactness of the particles and it can be used to detect changes in those properties under different conditions. Taking into account that some samples to be studied are not monodispersed, τ will be a function of the weight average of the molecular weight ( M w ) and of the z average of the dissipation factor (Qz) that depends on the size distribution of the particles:

τ = H M w Qz

(8)

From Eq. 8, it is possible to deduce a direct relationship between β and the diameter average of the particle [28]. Assuming an estimated value of -0.2 for γ for proteins in the range of 400 to 800 nm, β can be calculated from the slope of log τ vs. log λ curve, applying the following equation:

β = 4.2 +

∂ log τ ∂ log A = 4.2 + ∂ log λ ∂ log λ

where τ was measured as absorbance (A). Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

(9)

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Aiming to verify wether β was actually related to the average size of the particles, the size distribution functions and the hydrodynamic diameters of CAS particles were also determined by dynamic light scattering (DLS) with a He–Ne laser (λ0 = 632.8 nm) and 90° as the measuring angle.

A

Sapp fraction

1.0

0.8

0.6

0.4

0.5

B

τ (A650)

0.4 0.3 0.2

0.0 3.0

C

2.5 2.0

β

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0.1

1.5 1.0 0.5 0.0

0

10

20

30

-1

TCC (mmol L ) Figure 3. Mean experimental values (n=3) of fractional apparent solubility (Sapp) (A), turbidity (τ) as absorbance at 650 nm (B) and parameter β (C) in the supernatants of mixtures of ovine (●) and bovine (○) CAS (5 g L-1) as a function of the total calcium concentration (TCC), pH 6.8, temperature 25 ºC. The error bars representing the standard deviation for each data point (normal distribution of errors).

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María Eugenia Hidalgo, Manuel A. Mancilla Canales, Cássia R. Nespolo et al.

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The values of Sapp fractional change, τ and β for CCA suspensions in the supernatants were plotted (Fig. 3A, B and C respectively) as a function of TCC. For a given TCC, the Sapp of ovine CAS always reached lower values than the bovine CAS one (Fig. 3A). The average size of particles remaining in suspension, as estimated by β values, decreased during the precipitation as TCC was incremented (Fig. 3C). On the contrary, bovine CCA β values diminished at low TCC but rising again when both the precipitation and the amount of colloidal particles in suspension reached a limit value. Nevertheless, despite the shape of the β profiles it can be always observed that average sizes of the ovine CCA were higher, especially at low TCC values. As regards τ (Figure 3B) it increased up to a maximum at 20 mmol L-1 TCC in the case of bovine CAS and at 7.5 mmol L-1 TCC for the ovine CAS with a following clear decrease in both cases. For ovine CAS, β values underwent an abrupt decrease until ~5 mmol L-1 of TCC and then continued to decrease slowly to lower values (Figure 3C). These results would indicate the disassociation of the biggest CCA at high TCC. On the other hand, for bovine CAS, β values showed an initial formation of a low amount of quite small particles, followed by a second step with further formation of colloidal particles that progressively grew in size. As mentioned above, κ-CN represents a minority fraction in ovine milk in comparison with the bovine one suggesting an explanation to the higher average size of ovine CCA observed under all TCC. In addition, the hydrodynamic diameters determined by DLS showed a good linear correlation (r=0.9082; p35 CAG repeats) at the N-terminus of the Huntingtin gene causes HD, a fatal, autosomal dominant inherited neurodegenerative disorder characterized by motor and cognitive impairment along with personality changes. The resulting mutant huntingtin protein (mHtt) is prone to aggregate and form inclusion bodies in neurons of the striatum, though it has been indicated that aggregated mHtt may be a coping strategy against the more cytotoxic disaggregated conformation. mHtt may exhibit a toxic gain of function via its effects on glutamate production, chaperone proteins, and caspasses, among other factors [11,12]. Amyotrophic lateral sclerosis. Though the underlying cause of ALS remains unclear, familial forms of the disease are often associated with mutations along the SOD1 gene, which codes for Cu2+/Zn2+ superoxide dismutase, a cytoplasmic metalloenzyme involved in free radical neutralization [13]. Misfolded SOD1 has a disrupted dimer interface, unfolded beta barrel, and exposed hydrophobic core that make it prone to form amorphous aggregates [14,15]. Recently, the TAR DNA-binding protein gene has been implicated in non-familial forms of ALS, with TAR DNA-binding protein (TDP-43) as the major constituent of the ubiquitinated inclusions characteristic of ALS. Both forms of ALS present a toxic gain of function via non-amyloid, amorphous proteinaceous aggregates [16].

Protein Folding in Solution In general, a protein in solution has multiple conformational states, including its native state and unfolded state, in addition to intermediates between the two states. In the first stage of protein crystallization, we often observe protein crystals and protein aggregates. (Figure 2)

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Protein misfolding and aggregation a o occur both in vivo v and in viitro. When wee look into thhe process of protein aggregation, some intermediatess arise. As shoown in Figuree 3, protein m monomers and oligomers exxist in a state of o equilibrium m, with beta-shheet form inteermediates. Seeveral modelss for aggregaation have beeen postulated that all involve the formaation of an inntermolecular beta-sheet innitiated by am mino acid seqquences that act as nucleii for betaagggregation [1 17]. Under conditions c where the equuilibrium waas disturbed, oligomers prrogressed into o polymers orr profibrils, which w may evventually matuure into fibrils. There is evvidence to sug ggest that the oligomer o and profibril interrmediates are more m toxic thaan the final poolymer and fib bril products [7,18,19]. [

Fiigure 2. The purified p proteinn ERp44 was crystallized in 20 mM Tris––HCl (pH 7.5)) and 1.2 M diisodium succinaate at 16 ºC ussing hanging-drrop vapour difffusion (kindly provided p by CC C Wang and LK K Wang). We can c see soluble, crystal flocks, and turbid prootein. This sugggests that proteiins can have diifferent conform mational states in i solution undeer a certain condition. That is to t say, it is impossible for a prrotein to exist to otally in its natiive conformatioon in solution.

Many Factors Affect the t Prottein A Aggrega ation Prrocess oncentration. In general, a protein will precipitate p whhen its solubiliity value is Protein co exxceeded. Insullin is a major hormone invoolved in carbohhydrate, lipid and protein metabolism, m annd is critical to t the control of blood glucose level. Inn type I diabettes the effective level of innsulin is insu ufficient for normal n physioological functtion, and patiients must usse frequent innjections or in nfusions of innsulin. Severall tons of insuulin are produced annually for use by diiabetics. The tendency t for insulin i to agggregate and forrm fibrils has serious impliications for thhe production, storage and delivery of the protein, and a persists too be a probleem in new deelivery system ms currently under developpment. Thouggh a method has been developed to m minimize insullin fibrillationn [20], the moolecular mechanism of insuulin aggregatioon remains suubstantially un nknown. The current c hypothhesis offers thhat aggregationn arises from a partiallyfoolded intermeediate, with experiments e thhat attempt too correlate thhe presence of o such an inntermediate with w aggregatioon. The detailled mechanism m of aggregaation is being probed by deetermining thee regions of thhe molecule innvolved in the intermoleculaar interactionss leading to

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agggregation ussing various insulin i analoggs (point muutants), and innvestigating the t role of sooluble oligomeers.

Fiigure 3. Proteiin misfolding results r in aggrregation interm mediates, amylooid fibrils and amorphous agggregates. Hum man neuronal Tau T protein in the presence of o formaldehyde misfolded annd produced am morphous globu ule-like aggreggates (panels a,, b, and c). Huuman α-Syn misfolded m into amyloid-like a fibbrils (panels d, e, f, and g).

Ionic enviironment. Truumbell and Beckman B havee suggested ziinc deficiencyy to be the likkely linkage between b ALS’’s familial andd sporadic casees [21]. They show significcant change inn SOD1 reactiivity upon thee loss of zinc,, the absence of which alteers the redox state s of the Protein Aggregation, edited by Douglas A. Stein, Nova Science Publishers, Incorporated, 2011. ProQuest Ebook Central,

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remaining copper and overall stability of the protein dimer interface. The subsequent dissociation of zinc-deficient SOD1 into monomers may induce aggregation, a possible coping strategy against toxic insult to motor neurons by the lone copper. Mizorokia and coworkers establish that Al triggers Tau aggregation in vitro, though the group was unable to assess the effects in vivo due to Al-induced death of experimental animals before Tau aggregation might have be observed. Consequently, at high levels Al may pose a toxic threat to the body before reaching the brain, though this may not preclude the notion that Al may indirectly lead to AD following Al-associated organ deterioration [22]. The presence of certain ions has also been implicated in accelerating the aggregation process of α-Syn, the key pathological protein in PD [23]. Ultimately, much remains unknown about the role of ionic interactions in the protein aggregation pathway, but it has been proposed among other existing hypotheses that certain ions may deliver their cytotoxic effects role via the generation of ROS from hydrogen peroxide [5]. Chemical modification. Many types of modification are involved in the protein aggregation process with respect to neurodegenerative disease. Paired helical filament (PHF)/neurofibrillary tangles, the typical pathological feature of AD, consist of hyperphosphorylated Tau. Other post-translational modifications, such as glycation and sumoylation, were also found in the PHF-Tau [24-26]. Kuhla and coworkers have observed the promoting effects of reactive carbonyl compounds (RCCs) on Tau aggregation and filament formation [27]. α-Syn is the major component of the Lewy bodies characteristic of PD. Many research groups have found that chemical modifications such as glycation [28], sumoylation [29], and phosphorylation [30] are involved in α-Syn misfolding and aggregation. In recent years, researchers have also attempted to construct protein aggregation models using different chemicals. Glycation has been adopted by many researchers to modify neural proteins, such as Tau and α-Syn. Wei and colleagues employed ribose to treat BSA, establishing a model for globular amyloid-like aggregations [7]. Chen et al. obtained the amyloid-like aggregation of Tau and α-Syn by glycating them with ribose [18,19]. Here, we propose a putative mechanism for the formation of amyloid aggregates, whereby the chemical modification of native protein induces protein misfolding, and its eventual polymerization into amyloid fibrils (Figure 4). Formaldehyde, one of the most toxic organic compounds, is constantly being produced and degraded by cells of the human body. Nie and coworkers used formaldehyde to treat Tau protein in vitro, obtaining the amyloid-like aggregated Tau [31]. Unlike the typical globular protein BSA, these globular-like polymer deposits could be bound with the amyloid-specific dyes thioflavin T and Congo Red. These amyloid-like Tau aggregates were also found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells. Nie et al. also constructed a cell model for endogenous Tau aggregation using low concentration (0.010.1%) formaldehyde [32]. As shown in human clinical studies, endogenous levels of formaldehyde are kept relatively low (0.01-0.1 mM in urine) under physiological condition, but increase with age. At levels higher than that of the normal physiological concentration, formaldehyde induces misfolding of neural proteins such as Tau, resulting in cytotoxic products. Abnormally high levels of formaldehyde further leads to dysfunction of the central nervous system including cognitive impairment [33]. Furthermore, P25 and GSK3beta were up-regulated in the presence of formaldehyde added to SY5Y cell cultures using Illumina Solexa DNA sequencing, suggesting that abnormal formaldehyde concentration is related to

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hyyper-phosphorrylation. The urine u formalddehyde concenntrations of Allzheimer’s disease elders (nn=30) and norrmal elders (n= =30) (t=8.572, P