Modern Methods in Protein- and Nucleic Acid Research: Review Articles [Konferenzschrift, 1989, Bielefeld. Reprint 2019 ed.] 9783110853537, 9783110122756

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Modern Methods in Protein- and Nucleic Acid Research Review Articles

Modern Methods in Protein- and Nucleic Acid Research Review Articles

Editor Harald Tschesche

W G DE

Walter de Gruyter • Berlin • New York 1990

Editor Harald Tschesche, Dr. rer. nat. Professor für Biochemie Lehrstuhl für Biochemie Fakultät für Chemie Universität Bielefeld D-4800 Bielefeld Federal Republic of Germany

Library of Congress Cataloging-in-Publication Data Modern methods in protein- and nucleic acid research. Review articles Includes index. l.Proteins-Research-Methodology- Congresses. 2. Nucleic acidsResearch—Methodology—Congresses. I. Tschesche, Harald. QP551.M57 1990 574.19'245 90-14000 ISBN 3-11012275-8 (Berlin) ISBN 0-89925634-1

Deutsche Bibliothek Cataloging in Publication Data Modern methods in protein- and nucleic acid research : review articles / ed. Harald Tschesche. - Berlin ; New York : de Gruyter, 1990 ISBN 3-11012275-8 NE: Tschesche, Harald [Hrsg.]

© Printed on acid free paper which falls within the guidelines of the ANSI to ensure permanence and durability. © Copyright 1990 by Walter de Gruyter&Co., D-1000 Berlin 3 0 . All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Preface

This book complements the series of volumes on "Modern Methods in Protein Chemistry" published by de Gruyter in 1983, 1985 and 1988. It was the intention of this series to provide the growing family of researchers involved in the study of protein isolation, characterisation and modification with a compendium reviewing the latest developments in analytical and preparative methods. The aim was not to provide a complete listing of all efforts made in a particular field, but rather to give experts the opportunity to evaluate particular developments and to give a guide according to individual needs.

This is the first volume to include procedures required in nucleic acid chemistry and molecular biology. This is in accord with the intentions of the study group of the "Gesellschaft fur Biologische Chemie", which kindly supported the last conference on "Modern Methods in Protein and Nucleic Acid Analysis" held at the Centre of Interdisciplinary Research (ZIF) in Bielefeld in September, 1989. This volume contains some of the reviews presented during this conference, and additional reviewers were invited to present their methods in this book. Clearly there is a need for research laboratories involved in the study of proteins to have access to methods in protein and nucleic acid biochemistry as well as modern methods in molecular biology.

It is hoped, that this book will assist the reader in the evaluation and application of new methods in these fields and will help to avoid time-consuming and unnecessary errors.

Bielefeld, June 1990

Harald Tschesche

Contents Non-Radioactive Plaque and Colony Screening with Digoxigenin-ll-dUTP Tailed Oligonucleotides S. Schnierer, T. Gote, H. Tschesche

1

Oligonucleotide-Directed Mutation Construction W. Kramer, H.-J. Fritz

19

DNA Diagnostics - Applications and New Analytical Techniques B. S. Reckmann

37

Free Flow Electrophoresis for the Separation and Purification of Biopolymers H. Wagner, J. Heinrich

69

Separation of Proteins and Nucleic Acids using "Tentacle-Type" Ion Exchangers C. Jansen, W. Müller

99

Immobilized Metal Ion Affinity Chromatography of Recombinant Proteins E. Hochuli

117

Capillary Electrophoresis and Ion Spray Mass Spectrometry, New Powerful Methods for Separation and Characterization of Proteins and Nucleotides E. Bayer

133

Renaturation of Recombinant, Disulfide-Bonded Proteins from "Inclusion Bodies" R. Rudolph

149

"Activation Chromatography" - A New Approach for Dealing with Renaturation Problems in Protein Engineering Studies P. Flecker

173

Vili Progress in the Development of a C-Terminal Sequencing Method for Proteins and Peptides A. S. Inglis, F. Casagranda, B. Wittmann-Liebold

187

Determination of the Enantiomeric Composition of Amino Acids by Enantioselective Gas Chromatography W. A. König

213

Prediction of Peptide Epitopes in Proteins D. M. Dorati

231

Development of Specific Immunoassays for Thymosins W. Voelter, E. Livaniou, M. Mihelic

251

Bioaffinity Spin Labelling of Biomembrane Proteins R. I. Zhdanov

271

Cross-Linking between Nucleic Acids and Proteins A. Höckel, D. Prochnow, H. Fasold

297

Photoaffinity Labelling and its Combination with Biotin/Avidin Complexing in the Analysis of Peptide Hormone Receptors

305

D. Brandenburg, M. Fabry, F. Schumacher, U. Strack, F. Wedekind How Do You Get Large Proteins Crystals? Two Familiar Laboratory Methods Improved and Simplified G. Wiegand

343

Circular Dichroism as a Tool for Investigating Macromolecular Structure J. Reed

367

Modelling of Protein Structures on the Basis of Sequence Data

395

5. Suhai

IX

Analysis of Protein Structure in Solution by Two-Dimensional NMR Spectroscopy: 2D- 1 H NMR Investigation of Ribonuclease its Complexes with 2'- and 3'-Guanosine Monophosphates H. Rüterjans, E. Hoffmann, J. Schmidt, J. Simon Index

and

423

439

NON-RADIOACTIVE PLAQUE AND COLONY SCREENING WITH DIGOXIGENIN11-dUTP TAILED OLIGONUCLEOTIDES S. Schnierer, T. Gote and H. Tschesche

Introduction Several methods describe non-radioactive labelling of nucleic acids for use as hybridisation probes (1-5). The application of DNA hybridisation probes for detecting, localising and isolating specific nucleic acid sequences is well known. Some disadvantages surround the methods using radioactively labelled probes with

32

P: handling the radioactive material,

isotopic instability (short half-life) and long autoradiographic exposure times. Therefore, simple non-radioactive detection methods have become more popular. One of the most popular methods for labelling nucleic acids is the biotinylation of DNA by enzymatic incoproration of Biotin-ll-dUMP using standard nick translation (6) or 3'end labelling (7) protocols. Another popular method is chemical labelling of DNA via a photoactivatable biotin analogue using visible light irradiation. The labelled nucleic acids are recognised by an antibiotin antibody coupled with alkaline phosphatase and an enzyme-linked colour reaction. These methods gave satisfactory results of labelling DNA fragments

renaturation

step

f

( /

—

3),

(2)

SSG SSG

V (Eqn.

Y SSG SSG

formation

of

the

correct

disulfide bonds is catalyzed by GSH or other low molecular weight thiols (cysteine, cysteamine, 2-mercaptoethanol).

C The

GS-

Y

SSG SSG k

GSSG

v * GS" /y " yNGSSG

following

procedure,

X - — Y

V

/

as

SSG S-

modified

GS-

y

y GS-

from

; (26),

may

(3) be

utilized

for

disulfide bond formation via mixed disulfides: (1)

Incubate reduced, unfolded inclusion body protein at a protein concentration of 1-3 mg/ml in 0.05-0.1 M Tris-HCl, containing 0.1 M GSSG and-6 M GdmCl (or 8 M urea) at pH 9.3.

(2)

Incubate at room temperature for 5-18 h.

(3)

Quench derivatization by lowering the pH to pH 3 (e.g. by dropwise addition of 1 M HC1).

(4)

Separate mixed disulfide derivative from GSSG by dialysis (at 4°C against 0.1 M acetic acid or 0.01 M HC1) or by gel filtration (Sephadex G-25 with 0.1 M acetic acid or 0.01 M HC1 as eluant). Should the protein derivative precipitate, 6 M GdmCl (or 8 M urea) may be added to the dialysis/elution buffer.

163 (5)

Dilute mixed disulfide derivative into the freshly degassed renaturation buffer containing 0.05-0.1 Tris-HCl, 3-6 mM GSH (or other low molecular weight thiols) and 1 mM EDTA at pH 8-9. The protein concentration should be kept in the range of 5-100 /ig/ml.

(6)

Incubate at room temperature until renaturation is completed.

Renaturation

via

S-sulfonation, which may be used as an alternative

to

the formation of protein mixed disulfides, has been described elsewhere in considerable detail

(3,27,28).

Renaturation: Folding vs. Aggregation a) Dependence on protein concentration

«mm y

Figure

3:

Schematic illustration of the kinetic competition between proper folding (A: formation of a folding intermediate; B: formation of the native structure) and aggregation (C: aggregation of unfolded polypeptide chains; D: aggregation by incorrect domain interactions)

164 Upon

protein

disulfide

renaturation,

bond

formation)

with the formation

the has

pathway been

of inactive

of correct refolding

found

to

aggregates

be

in

kinetic

(29). As

(including competition

illustrated

in

Fig-

ure 3, aggregation may originate from non-specific (hydrophobic)

interac-

tions

as

at

the

level

incorrect domain

of the unfolded polypeptide

chain,

as well

interactions of partially structured folding

ates (30). In both cases, second (or higher) order aggregation compete with first order folding steps. Upon increase of the

intermedireactions concentra-

tion of unfolded polypeptide chains, the half-time of the folding tions

remains

constant,

while

the

rate

of

aggregation

from

reac-

increases.

Consequently, aggregation predominates during renaturation above a limiting concentration served

at

described

of denatured protein. The increase

increasing by

a

concentrations

simple

competitive

of

denatured

(folding

vs.

in aggregation

polypeptides aggregation)

may

obbe

reaction

mechani sm:

native

denatured

(4) aggregates

On the basis of the given kinetic model, the yield of reactivation can be computed as a function of the initial concentration of denatured

protein

(Eqn. 5).

y

= 100 • kj D0

y:

• K2

.in

(i + Dp • k 2

)

(5)

kj

yield of native protein (%)

DQ: molar concentration of denatured protein present at t = 0 kj: rate-determining folding step (1st order) k 2 : rate-determining aggregation step (2nd order)

165 Using this model, the decrease in the yield of reactivation of denatured lactic dehydrogenase, which has been previously determined as a function of Dp

(29), can be calculated with a normal

folding rate and diffusion

D 0 (J*) Figure 4: Effect of the concentration of denatured protein (DQ) on the extent of reactivation of porcine muscle lactic dehydrogenase (cf 29). Full lines are calculated according to eqn. 5 with M kj = 0.1 s"l and k2 = - s ~ l . Molar concentrations are based on a subunit molecular weight of 35 kDa. The

preponderance

of

aggregation

at

higher

protein

concentrations

has

been a major obstacle for the commercial application of in vitro folding processes.

Renaturation

at low protein concentrations would

necessitate

large reaction volumes, resulting in high production costs. This problem can be circumvented by a properly controlled renaturation procedure. Provided

that

below

a

the

actual

critical

level

concentration where

of denatured

aggregation

does

polypeptides not

is

kept

predominate,

high

yields of renaturation per volume of solution can be obtained (31). This may be achieved by slow and continuous, as well as by discontinuous addition of denatured protein into the renaturation solution (Figure 5).

166

e >>

> aa

the B o w m a n - B i r k - t y p e amino

subdomain

proteinase

acid replacements are

given.

in

the

175

by

denaturation

under

reducing

conditions

and

subsequent

renaturation. In Wettlaufer's system a mixture of reducing and oxidizing agents of defined ratio is used to facilitate the slow reshuffling of incorrect disulfide bridges [5]. The renaturation problem is even more limiting with artificial proteins.

Throughout protein engineering

attempts of de novo protein design

studies

[6] and

all

[7-8] deleterious refolding

properties are expected for protein variants, whose amino acid sequence has been changed by site-directed mutagenesis especially in those positions required for attaining a correct pathway of refolding. Here, a new approach to deal with such difficulties, 1.e. using a protein affinity ligand as a matrix with complementary structure, is described. 2.

Material and Methods

All materials and procedures, details of expression,

cyanogen

bromide cleavage, the refolding protocol for the parent Bowman Birk type proteinase inhibitor and the procedure for assaying residual trypsin and chymotrypsin activities have been published [9,10]. Bovine pancreatic trypsin was obtained from Boehringer Mannheim

and active

Sepharose [12].

5,83.10"

[11]. Trypsin

4B was prepared according to a published

The 9

site titrated as described

final

concentration

of trypsin

M. Bovine chymotrypsin,

in the

procedure assays

was

containing a minimum of 95%

active material, and the synthetic substrate were obtained from Sigma. The final chymotrypsin concentration in the assays was 4,16.10"8 M. The amounts of inbhibitor were calculated by the expressions

I = [E°] . V. Mr/R

and

R = Veq/Vtot.

176

[E°] is the final concentration of proteinase in mol/1, V the final

volume

of the

enzyme

assays

(6.10~41), Mr the

molecular mass of the inhibitor (8.109 ^.g/mol),

relative

the volume of

the aliquot of diluted stock solution at the equivalence point and Vtot the volume of diluted stock solution corresponding to the total amount of inhibitor. The determination of the equivalence points of the titrations has been described in detail [10]. Refolding/affinity

chromatography

for

mutant

proteinase

in-

hibitors Method A: 32 mg crude cyanogen bromide cleavage mixture were incubated in 240 (J.1 of reduction/denaturation buffer, containing 2 M Tris/Cl (pH 8), 0.2% EDTA, 6M guanidinium hydrochloride and 2 M 2-mercaptoethanol at 37°C for 18 h and added dropwise to 240 ml of renaturation buffer containing 80 mM Tris/Cl

(pH 8), 0.1

mM EDTA and 0.2 mM oxidized glutathione at 37°C under vigorous stirring. The refolding mixture was shaken for 60 min at 37°C, trypsin-Sepharose was added and shaking was continued at 32°C for 90 min. The matrix was transferred to a column and washed with 50 ml 0.1 M sodium borate (pH 7.6) containing 10 mM CaCl2, 50 ml 10 mM CaCl2 and 10 mM HC1 in the case of the P^-Pro and 5 mM HC1 in the case of the P2'-Pro and the P3'-Leu variant at 4°C. The

P/ -Pro

variant

was

eluted

with

100

mM

HC1,

the

other

variants with 50 mM HC1 at a flow rate of 0.2 ml/min. fractions were collected and 80 (J.1 aliquots were assayed

5 ml for

residual trypsin activities. The active fractions were pooled, diluted with the same volume of water and lyophilized to dryness.

Method B: The refolding mixtures were incubated for 6 h at 37°C and concentrated by repeated ultrafiltration to remove all low molecular weight compounds as described

[9], The samples were

dissolved in 15 ml water and applied to trypsin-Sepharose at 4°C

177

at a flow rate of 2 ml/min for 2 h. The respective variants were eluted as described in Method A. The combined flow-throughs of each experiment were concentrated and desalted by ultrafiltration, lyophilized and titrated with trypsin and chymotrypsin. The samples give single bands at 8 kDa of SDS gels and single active peaks on reversed-phase HPLC. The retention times are in accordance with the parent protein [9]. 3.

Results

In the case of the parent inhibitor, the isolated yield after the reduction/denaturation, the subsequent refolding step affinity chromatography (the yield of this step is greater than 95% in our hands),

is at a maximum at 30%

[9]. According to Fig.

1, a

stoichiometric ratio of 1:1 between the trypsin reactive and the chymotrypsin reactive subdomain is expected for this protein. Comparative titration (a simple test, which takes a half day of laboratory work in our hands) gave a stoichiometric ratio of 1:1 between both subdomains in the case of the parent inhibitor. This ratio remained constant within an experimental error range of +/10%, both in the crude preparation obtained by renaturation and ultrafiltration (Fig. 2A/1) on the one hand, and after subsequent affinity chromatography on trypsin-Sepharose

(Fig. 2B/1) on the

other. In contrast, the single amino acid replacements (constructed to test specific questions on the mechanism of proteinase inhibitors [13] and not occurring in other members of the Bowman Birk family of proteinase inhibitors [14]) P/Ser

—

>

Pro

P2' Asn

—

>

Pro

P3' Pro

—

>

Leu

and

178

1

Fig.2.

Results bars)

of

2

3

titration

4

experiments

and c h y m o t r v p s i n ( h a t c h e d

protein;

2:

the

P^'Pro-;

3:

with

trypsin(fi1led

b a r s ) . 1:

the

P2'Pro-;

the 4:

parent the

P3'Leu-variant. A:

yields(in

bitory

pg)

subdomain

of

trypsin-

and

chymotrvpsin-

before

the

contact

yields(in

pg)

after

with

inhi-

trypsin-

Sepharose. B:

respective

tography

on

the

matrix.

activation

chroma-

179

resulted in dramatic deviation from a stoichiometric ratio of 1:1. The results of the titration experiments of crude preparations are given in Fig. 2A/2-4. The relative amount of trypsin reactive material is reduced to a small fraction of the chymotrypsin reactive subdomain in the case of the P/Pro- and the P3'Leu-variant. activity.

All

The

P2'Pro-variant

attempts

to

is

achieve

devoid

better

of

any

trypsin

renaturation

by

a

systematic variation of temperature [15], the time of refolding, the dilution [9, 16], the ratio of B-mercaptoethanol and oxidized glutathion [16], the addition of structure stabilizing ions [17] and the presence of thioredoxin from Corynebacterium nephridii to facilitate reshuffling of incorrect disulfide bridges

[18]

remained unsuccessful. Nevertheless, a stoichiometric ratio of both subdomains was induced into these variants by the presence of trypsin-Sepharose either by the addition of trypsin-Sepharose to

the

refolding

mixture

(Method

A)

or

by

simple

affinity

chromatography after renaturation and ultrafiltration (Method B) . The absolute and relative increase of the amount of the trypsin reactive domain in the variants is seen on comparison of Fig. 2A/2-4 and 2B/2-4. This is in contrast to the situation in the parent protein displaying the same correct ratio of 1:1 before and after affinity chromatography. The accuracy of the titration experiments in the crude preparations was confirmed by addition of known

amounts

proteins

and

chymotrypsin

of correctly

redetermination inhibitory

refolded mutants to the of

material.

the

amounts

Clearly

the

of

crude

trypsin

and

stoichiometric

ratio of both subdomains achieved in the presence of trypsinSepharose is not the result of simple affinity chromatography. In this case, the minor amounts of trypsin reactive material will be retained on the column together with the same minor amount of chymotrypsin reactive material. However, no trace of chymotrypsin inhibitory activity was detectable in the combined flow-throughs

180

of each experiment after concentration and desalting by ultrafiltration . The

rate

of

refolding

of the

trypsin

reactive

subdomain

in

comparison to the chymotrypsin reactive subdomain is much slower in the P/Pro-variant in comparison to the parent protein. The variants

eluted

from the

affinity

columns

were

subjected

to

equilibration experiments in the refolding buffer for prolonged times

and

assayed

activities. decreased

for

trypsin

The relative amount

slowly

towards

the

and

chymotrypsin

inhibitory

of trypsin reactive

low

initial

level

material

(unpublished

observations in our laboratory). 4.

Discussion

The strong deviation

from a stoichiometric

ratio between the

subdomains points to significant structural irregularities in the variants.

These effects are localized to the same

subdomain,

which has been subjected to single amino acid replacements. A major difficulty of protein engineering studies is to detect and control refolding problems in a protein, which has been modified by site-directed mutagenesis. Therefore, a clear-cut dissection between structural and functional mutants [9, 10] is of critical importance for an unambiguous interpretation of the results. The Bowman Birk

inhibitor of serine proteinases proved

itself an

excellent model protein for this purpose. The incorrect amount of trypsin reactive material in the variants is shifted to correct values by the presence of trypsin-Sepharose as a matrix with complementary structure. It is conceivable, that such

an

affinity

matrix

should

be

effective

in guiding

the

refolding process of a protein variant towards the conformation of the native protein. Similar observations on the activation of

181

recombinant

subtilisin by the external

sequence were published

recently

addition

[19] and point

of

its Pro-

in the

same

direction. To the best of our knowledge, our paper [10] was the first report

on the use of an affinity matrix

for

mutant proteinase inhibitors. The term "Activation

refolding Chromatog-

raphy" is proposed for this new approach. The stability of the variants, both in the elution step (50-100 mM HC1)

and on equilibration

in the refolding buffer,

is in

accordance with a high energetic barrier between the folded state N and N*, the transition state of unfolding as postulated by Creighton [20] (Fig. 3). In the case of a structure stabilizing effect

(in a thermodynamic

sense)

exerted

by

the matrix,

a

subsequent equilibration back to the initial amounts of trypsin reactive material had to be exprected for the variants. The slow equilibration

is in accord with a kinetic effect of trypsin-

Sepharose on the refolding process and independent support for the cardbox protein folding model of Goldenberg & Creighton [21]. The transition state (N*) of the folding reaction as a distorted form of the folded state

(N) is apparently

stabilized by the

matrix resulting in a combined action of kinetic and thermodynamic effects on the protein folding process of the trypsin reactive subdomain. The failure to obtain a ratio of 1:1 in the titration experiments with free trypsin points to a specific stabilization of N* by distorted trypsin molecules being present on the solid matrix only. Fine distortion of immobilized proteins are known [22] and could be a crucial factor for the success of further

experiments

with

similar

systems.

In

any

case,

the

renaturation of the variants under non-reducing conditions by Method B suggests that the transition

from N to N* does not

concur with rearrangements of disulfide bonds (although this is no definite proof, as traces of active SH-groups might still be present after ultrafiltration or in incorrectly folded intermediates) .

182


0l?0P0CH2 AcJe HO HO OH U-O-j 0.0 o=c

0 0 0 ii ii ii HOfOfOlfOCH

I* 0 V 0 0 0 r~\ ii II II D-N V-OPOPOP OCH- Ad HO HO OH L'O

A(je

HO HO OH L^O-vJ

HO OH

8 o=c0

NH, 0 0 0 HOf>CH2!foi)oCH2 A c j e HO O OH L-'O-vj 10

0 0 0 II II II HOI^O^OPOCHj HO HO OH

o,o o=c

11

3

n-^nr

ft

0,0

0=C-t=

Figure 7. Spin-labeled ATP analogues. ATPase (36). In this study we were interested in the possibilities of using SL-ATP analocrues for the probing of rabbit sarcoplasmic reticulum

(SR) Ca-ATPase. Ca-pump is being studied for

a long time (37), and now the Ca-ATPase structure is comparatively well-known: the primary structure (38), the localization of Ca-binding and phosphorylation sites (39) as well as Ca-channel (40). Nevertheless the structure-functional studies of CaATPase by spectroscopic techniques including spin labeling are of considerable interest

(41-44).

283

Synthesis The synthesis of SL-ATP £ was reported by two research groups (27,28). We modified its synthetic procedure for the synthesis of SL-ATP (45) . This technique was later published by Repke et al (36) and in Russian monograph (31). According to this procedure (31,36,45) the 4-fold excess of equimolar mixture of 3-carboxy-2,2,5,5-tetramethylpyrrolidinoxyl(46) and N,N-carbonyldiimidazole is added to ATP 30 min after its preparation. The nucleotides were precipitated by the excess of acetone after 4 5 min. The yellow precipitate was dried and applied on silica gel column. The elution of spin-labeled ATP was carried out with n-butanol/water/acetic acid (5:3:2) mixture. The fractions containing SL-ATP (EPR control) were extracted with ether after the addition of water. The water layers were evaporated, and the residue was lyophilized. The yield of SL-ATP 1_ is 31%. R,=0.35 (PEI-cellulose, 0.4 M KH-PO., pH 3.5) f ' 2 4 ' ^ ' '•"'max 257 nm (£ 14900); a =16.1 G(water); adenin:phosphate=0.35:1.0.

SL-ATP Substrate Activity SR vesicles were prepared from the white skeletal muscle of rabbit hind legs (47,48). According to electrophoresis and electron microscopy data the Ca-ATPase preparation is represented by membrane bubbles 95 per cent of protein being Ca-ATPase (the phospholipid content is equal to 0.7 mcmol P^mg (43,48). This Ca-ATPase preparation was characterized by the rate of P. -

1

-

1

1

production equaled to 13 mcmol mg min (48), but the velocity of the hydrolysis of SL-ATP 2 with Ca-ATPase was 2 mcmol mg min . However, SL-ATP analogue 6 containing pyrroline nitroxyl moiety was not hydrolized by the same Ca-ATPase preparation (44) . SL-ATP 1_ is also hydrolized by myosin Ca-ATPase from skeletal muscle. Myosin preparation was isolated by using method previously described (49,50). Fig.8 presents the Lineweaver-Burk

284

Figure 8. The Lineweaver-Burk plot for the hydrolysis of SL-ATP 2 with myosin Ca-ATPase (10). Ca ions were introduced into solution in the form of C a C ^ . The concentration of Ca -SL-ATP was variated from 2 0 mcM to 2 mcM. Conditions: 0.6 M KC1; 2 0 mcM myosin; 50 mM Tris-HCl, pH 7.5; 25°C. plot for the hydrolysis of SL-ATP with myosin. While the myosin Ca-ATPase activity during the ATP hydrolysis was 36 mcmol P.- 1 - 1 -11 g min , for the SL-ATP hydrolysis it was 114 mcmol P^g min + ^ (10). The Michaelis constant, K s equals to 30-20 mcM and values 60-40 mcM for ATP and SL-ATP —7, respectively. The V^ _1 _1 ' are equal to 0.25 meg sec and 0.3 meg sec , respectively. It follows from these data that myosin has much more affinity to SL-ATP as compared with ATP. The interaction of SL-ATP and manganese ions was also studied to testify the bioactivity of SL-ATP

(51).

EPR Measurements EPR spectra were recorded on Varian E-109 radiospectrometer with variable-temperature control system (-1°C) in the flat quartz cell (100 mcl). To prepare the samples for recording

285

EPR spectra, aliquots of concentrated solutions of SL-ATP, ATP, MgC 12, CaC]_2, NiCl 2 , K 3 Fe(CN) 6 and EGTA were added, as required, to 9 mg/ml SR Ca-ATPase to form a final volume of 100 mcl and 8 mg/ml ATPase. Modulation amplitude of 0.1 G, time constant of 0.1 sec and microwave power of 2 mW were used in all cases. The values of correlation time TT for SL-Ca-ATPase were calcuc latedfrom the equation (52) (Fig.9): < T c = 6 -65 where

H+1

" 1) 10" 1 0 sec

( V I + 1 / I_1

is the peak to peak line height of the EPR low-

field line, I + 1 and

are the amplitudes of low-field or

hiah-field components of SL-ATPase EPR spectrum. To determine the parameters of rotational diffusion for the SL-ATP-Ca-ATPase complex the Tf

values were calculated as an average quantity

from 15 measurements of various Ca-ATPase preparations using statistical analysis.

40 1

--19.0

1—

20 i

i

0 •

t°C

--21.0

- -23.0^ /f

1

/ r

««3

T 3.1

3.3

3.5

3.7

Figure 9. The Arrenius plot for the correlation time^ET, of SL-ATPase EPR spectrum (10,43). Conditions: 8 mg/ml (0.08 mM) SR Ca-ATPase, 0.17 mM MgCl 2 , 0.3 mM EGTA, 0.09 mM SL-ATP 7, pH 6.8, 25°C.

286

The I + .J -component of spin label isotropic EPR spectrum was used as SL-ATP concentration measure for reduction rate calculations The reaction rate of nitroxyl reduction with sodium ascorbate is circumscribed by the equation (K - reduction rate constant): V = -d I + 1 / dt = K /A/ /R/, where is the amplitude of low-field EPR component, t -time, /A/ - the ascorbate concentration, /R/ - spin label concentration. The plots were presented as the time dependence of In values. The K values can be estimated from the angle of the slope of these curves. After usual transformations the equation can be obtained ( At is a time interval ) : K = A In I+ 1 / A t /R/, which was used to calculate the reduction rate constants from the kinetic plots ( for the review see, i.e. (53). The kinetic data analysis was carried out with the use of the logarithmic extrapolation method and PC ISKRA (KURSK). So called "paramagnetic probe - spin label" technique (54) was used to determine the exchange relaxation rate constants for the interaction of SL-ATP with negatively (ferricyanide) or positively (Ni++) charged paramagnetic ions. These constants, K , were calculated from the equation: exch ^ A A

H +1^ = 6 . 5 10~8 K exch , C , '

where a a H + ^ is the difference between the AH + ^ values after and before the addition of paramagnetic ions, C - the concentration of paramagnetic ion, M.

Ca-ATPase probing The line shape and the correlation time values of Mg++-SL-ATP 1_

287

triplet spectrum with and without Ca-ATPase are very close. While the TT value without enzyme equals to 0.90-0.10* 10 ^ ^ + — 1 0 sec, the Cys and Thr-89 —> Ser destabilized conformation and reduced ATP-binding simultaneously. Although position 89 may be at the ATP-binding site, position 17 is probably not; they therefore suggested that affinity for ATP was reduced as a result of general changes in conformation affecting the binding site. The contribution of histidine-1 to the conformation and activity of glucagon was reviewed by Flanders et al. (34). They found an unexpected effect of substituting Phe, Ala or desHis at position 1 was a marked increase in helicity, probably due to increased trimerization, and pointed

378 out that conclusions based on changes of activity following a single amino acid substitution could follow from long range conformational effects as well as specific chemical differences in the side chain. Finally Epand and Liepnicks (35) carried out chemical modification of Arg-17 and Arg-18 in glucagon in order to increase solubility. They used CD to show that the modified form had the same conformation as native glucagon in dilute solution as well as the same ability to activate adenyl cyclase, so that it could serve as a more convenient experimental material. A vast amount of work has been done using CD to follow protein unfolding and refolding as a function of pH, temperature, denaturing agents or solvent changes (36-48). The ability CD offers to observe total secondary structure directly presents a distinct advantage in folding studies over methods based on the observation of local probes. Because of its extreme sensitivity in measuring slight differences in secondary structure content, one can follow subunit association/dissociation (36,48) as well as the folding and/or unfolding of individual protein chains. CD can also serve to establish the integrity of native conformation after modification, as seen earlier (35). Lin et al. (38) used both far UV and near UV CD to demonstrate that introducing an extrinsic cross-link between Lys-7 and Ly-41 in ribonuclease A, performed to aid in folding studies, had no significant effect on the secondary structure content or tyrosine CD of the native enzyme. Finally, and most simply, far UV CD can serve to establish the total secondary structure content of a native protein and so obtain some idea of its conformation when X-ray crystallography or NMR cannot be used. On their own, the data for percent a-helix, P-sheet and remainder may be of limited value, though useful for comparative studies (49-55) between similar proteins in different species or families of proteins functionally similar but sequentially distinct. But CD-derived secondary structure data may be combined with other physical measurements to construct a fuller structural model of the protein in question. Lane and Jardetzsky (56) combined CD with 'H-NMR to posit a preliminary structural analysis of the trp repressor. Siezen and Argos (57) made predictions of lens crystallin secondary structure derived from folding algorithms and checked the results with CD. Shin and McFarlane (58) put together results from secondary structure prediction methods, far UV CD and 'H-NMR to infer certain properties of the tertiary structure of myelin P2 protein. Peptide far UV CD -Interpretation. Although most peptides are small enough to allow full structural analysis from 'H-NMR, it is sometimes more convenient to use CD if

379 either the supply of peptide or access to a > 400 MHz NMR is limited. Woody (5) has published an extensive review of peptide CD, discussing the peptide chromophore CD, aromatic and disulfide contributions as relevant to the interpretation of peptide dichroism. As mentioned before, in estimating secondary structure content from far UV CD of peptides, one is better off using basis spectra of model peptides in standard conformations and fitting the curve linearly than using methods based on globular proteins either directly or indirecdy. Although the intensity of the bands for pure helical model peptides can vary with the side chain (Fig. 3), the variability principally affects the low wavelength positive maximum and is sufficiently small throughout so that predictions of helix content in heteropolypeptides are fairly accurate. The CD spectrum for (J-sheet is much more dependent on side chain (Fig. 4) in both amplitude and position of the bands, although it does appear to be a single spectral type. An attempt has been made by Fasman and his group (59) to divide the spectra observed into a 'normal' I-(3 type and a II-p type where the extrema are red-shifted, possibly due to the presence of (3 turns. Despite these drawbacks, basis spectra for (3 sheet from model peptides function sufficiently well to be in general use. 100

Figure 3: Effect of side chain on the intensity of the CD signal from a-helix. Poly-L-glutamic acid (in water, pH 4)

, poly-L-

lysine (in water, pH 11)

,

5

poly-[N -(2-hydroxyethyl)-L-glutamine (in 8:2 methanol:water)

,

poly-L-alanine (in TEE:TFA 98.5:1.5) Spectra from Adler et al. (9).

-50

180

200

220

240

260

380

Figure 4: Effect of side chain on the intensity of the CD signal from p-sheet. Poly-L-lysine

,

poly-(Lys-Leu-Lys-Leu) poly-L-serine

,

, poly-5-

carboxymethyl-L-cysteine Redrawn from spectra in Refs. 11, 5,61 and 9, respectively). The greatest problem in the use of model peptides to produce basis spectra for estimating secondary structure comes from selecting suitable models for random coil and for p-turns. In the first instance, part of the difficulty comes from confusion about the definition of "random coil". A majority of model peptides show a particular type of highly similar spectrum under conditions designed to disrupt formation of regular hydrogenbonded structure. The term "random coil" used for this is a little misleading in that, although meant to signify the absence of regularly repeating elements, it tends to imply that the peptide is freely rotating around all N-C a and C a -C' bonds. As demonstrated by Aebersold and Pysh (60), the calculated CD spectrum for an octapeptide freely rotating through -180«J>