Chemistry of peptides and proteins: Vol. 1 Proceedings of the Third USSR - FRG Symposium, Makhachkala (USSR), October 2–6, 1980 [Reprint 2019 ed.] 9783111415086, 9783110086041

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Chemistry of Peptides and Proteins Volume 1

Chemistry of Peptides and Proteins Volume 1

Proceedings of the Third USSR—FRG Symposium Makhachkala (USSR), October 2-6,1980 Editors Wolfgang Voelter • Erich Wünsch Yuri Ovchinnikov • Vadim Ivanov

W G DE

Walter de Gruyter • Berlin • New York 1982

Editors: Wolfgang Voelter, Prof. Dr. rer. nat. Director Department of Physical Biochemistry Institute for Physiological Chemistry Hoppe-Seyler-Strasse 1 D - 7 4 0 0 Tübingen • Germany Erich Wünsch, Prof. Dr. rer. nat., Dr. med. h.c. Director Department of Peptide Chemistry Max-Planck-lnstitute for Biochemistry D - 8 0 3 3 Martinsried • Germany Yuri Ovchinnikov, Prof. Dr. Vadim Ivanov, Prof. Dr. Director Shemyakin Institute for Bioorganic Chemistry USSR Academy of Sciences Moscow • USSR CIP-Kurztitelaufnahme der Deutschen Bibliothek

Chemistry of peptides and proteins : proceedings of the . . . USSR-FRG symposium. - Berlin ; New York : de Gruyter Vol.1. Proceedings of the third USSR-FRG symposium : Makhachkala (USSR), October 2 - 6 , 1 9 8 0 . - 1982. ISBN 3-11-008604-2

Library of Congress Cataloging in Publication Data

USSR-FRG Symposium (3rd : 1980 : Makhachkala, R.S.F.S.R.) Chemistry of peptides and proteins. Bibliography: p. Includes index. 1. Peptides-Congresses. 2. Proteins-Congresses. I. Voelter, W. II. Title. QD431.A1U88 1980 547.7'5 82-14937 ISBN 3-11-008604-2

Copyright © 1982 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

PREFACE The USSR-FRG Symposium on the Chemistry of Peptides and Proteins was conducted to reinforce between scientists of both countries the hitherto existent contact and exchange of information at the level of the European Peptide Symposium. Based on the scientific agreement between the Union of the Soviet Socialist Republics and the Federal Republic of Germany, represented on the one hand by the Soviet Academy of Sciences and on the other by the Deutsche Forschungsgemeinschaft, the bilateral symposium series was initiated at the 1st Meeting in Dushanbe-Tadjikistan in April, 1976. As the scientific basis of these meetings, we have attempted to combine the three main research areas in the field of chemistry of peptides and proteins, i.e. a) isolation, b) structure elucidation and c) synthesis as a discussion platform in order to reestablish the working community previously existing in chemistry of natural products. The first meeting has already confirmed the usefulness of such a programme . Additionally, our idea was to incorporate, if required, discussions on special, closely-related topics of particular scientific interest. Consequently, at the 2nd Meeting in Grainau-Eibsee in May, 1978, membrane chemistry was added to the programme. The positive aspects of this choice were clearly confirmed at the 3rd meeting in Makhachkala in October, 1980, where a first, tentative discussion on immunological problems in connection with the chemistry of peptides and proteins took place. We believe that this widening of the scientific programme may, in future meetings, lead to a fruitful exchange of experience and knowledge in the individual sectors of this field of chemistry. The strong resonance of the scientific communications and related discussions among

VI

the participants of both countries is clearly shown by the increasing interest in these meetings and by the continuous demand for publication of the reports. Following a simple abstract booklet on the occasion of the 1st Meeting, a proceedings volume with shortened versions of the lectures was printed for the 2nd Meeting. In the present proceedings the detailed reports of the lectures and communications of the 3rd Symposium on the Chemistry of Peptides and Proteins are published in full. The organizers of these series of symposia on Chemistry of Peptides and Proteins would like to take the opportunity of expressing their gratitude to the Soviet Academy of Sciences as well as to the Deutsche Forschungsgemeinschaft for their generous support and sponsorship.

For the Editors E. Wünsch

VII

CONTENTS

I. Isolation and Structure Elucidation of Peptides and Proteins The Primary Structure of DNA-Dependent RNA Polymerase from E.coli. Nucleotide Sequence of rpoB Gene and Amino Acid Sequence of the B-Subunit V.Lipkin, G.Monastyrskaya, V.Guvanov, S.Guryev, 0.Chertov, V.Grinkevich, I.Makarova, T.Marchenko, 1.Polovnikova, E.Sverdlov, Y.Ovchinnikov

3

Study on the Primary Structure of the Elongation Factor G from E.coli Y.B.Alakhov, L.P.Motuz, N.V.Dovgas , Y.A.Ovchinnikov

13

Intracellular Serine Proteases of Bacilli A.Ya.Strongin, V.M.Stepanov

19

Structure and Biological Function of Proteinase Inhibitors from Yeast K.Maier, H.Holzer, R. Barth ,P . Biinning , E.Kominami

29

Inhibitors of Human Neutral Granulocytic Proteinases from the Leech: Biochemical Characterization and Pathobiochemical Aspects U.Seemuller, M.Eulitz

39

Isolation of a Specific Protein Inhibitor of Fungal Proteinase and Yeast Proteinase B from the Kidney Bean Seeds V.V.Mosolov, E.L.Malova, A.N.Tcheban,V.M.Lakhtin , A.N.Bakh

47

Isolation of Membrane Glycoproteins from Paramyxovirus SV5 for Analysis of their Carbohydrate Structure P.Prehm

53

Haemoglobin Polymorphism in Chironomus(Diptera) H.Aschauer, T.Kleinschmidt, W.Steer, G.Braunitzer

61

High Altitude Respiration of the Bar-Headed Goose (Anser Indicus) and the Different Evolution of thecC- and 6-Chains in Avian Haemoglobins G. Braunitzer, W. Oberthiir

71

VIII T h e S t r u c t u r e of the D i c y c l o h e x y l c a r b o d i i m i d e Binding Subunit E.Wächter,T.Graf, G.Wild, W.Sebald

83

Structural Studies of the Active Ion Trans-port Systems N . M o d y a n o v , A . B a b a k o v , N . A r z a m a z o v a , K. Dzhandzhugazyan, S.Kocherginskaya

85

I s o l a t i o n of t h e T T X - S e n s i t i v e P r o t e i n ( S ) of E x c i t a b l e T i s s u e s V.K.Lishko, M.K.Malysheva, A.V.Stefanov,A.M. Chagovetz, A.A.Bogomoletz

93

Fragments Formed from Neuropeptides of H y p o t h a l a m i c E n d o p e p t i d a s e s T.Akopyan, A.Arutunyan, A.Oganisyan

99

upon

Action

N e w D a t a a b o u t the S p e c i f i c P r o t e i n s of H y p o t h a l a m u s - C a r r i e r s of C a r d i o a c t i v e C o m p o u n d s A.Galoyan, R.Srapionian

103

I s o l a t i o n a n d S t r u c t u r a l S t u d y of N e u r o t o x i n f r o m t h e V e n o m of S p i d e r L a t r o d e c t u s T r e d e cimguttatus S . S a l i k h o v , M . T a s h m u k h a m e d o v , M . A d y l b e k o v , J. Abdurakhmanova, A.Korneyev, A.Sadykov

109

Sea Anemone L.Beress

121

Toxins,

a

Minireview

S t r u c t u r e a n d P r o p e r t i e s of M a s t o p a r a n II O l i g o p e p t i d e from the V e n o m of H o r n e t V e s p a orientalis I.Nasimov, L.Snezhkova, O.Reshetova, A.Miroshnikov

127

Isolation, Physico-Chemical and Biological Prop e r t i e s of t h e I m m u n i t y P o l y p e p t i d e B i o r e g u l a t o r from Thymus O.A.Pisarev, V.G.Morosov, V.K.Khavinson, L.K. Shataeva, G.V.Samsonov

137

Some Recent Advances in the M e t h o d s of Protein Sequence Analysis A.Henschen, F.Lottspeich, W.Brandt, C.v.Holt

143

Field Desorption Mass Spectrometry peptides W.Voelter, M.Przybylski

153

of

Oligo-

IX S e q u e n c e A n a l y s i s of M e m b r a n e - M o d i f y i n g P e p t i d e Antibiotics by Gas Chromatography and Mass Spectrometry W.A.König, M.Aydin

173

S t r u c t u r e E l u c i d a t i o n and P r o p e r t i e s of N i k k o m y c i n s , a N e w C l a s s of N u c l e o s i d e - P e p t i d e A n tibiotics H.Hagenmaier, W.A.König

187

II. P e p t i d e S y n t h e s e s . B i o l o g i c a l A c t i v i t y a n d A n a l y t i c a l P r o b l e m s of S y n t h e t i c P e p t i d e s

Electrochemical Introduction and Selective Rem o v a l of a N e w T y p e o f A m i n o - a n d C a r b o x y - P r o tecting Group for Peptide Synthesis G.Jung, M.H.Khalifa, A.Rieker

193

N e w A s p e c t s of P e p t i d e S y n t h e s i s b y F o u r C o m ponent Condensations I.Ugi, W.Breuer, P.Bukall, S.Falou, G.Giesem a n n , R . H e r r m a n n , G.Hiibener, D . M a r q u a r d i n g P.Seidel, R.Urban

203

Isocyanides as Activating Reagents Synthesis H.Aigner, G.Koch, D.Marquarding

2 09

Impact of C o n f o r m a t i o n on the gies for Peptide Sequences M.Mutter, H.Anzinger, K.Bode, Pillai

in

Peptide

Synthetic F.Maser,

StrateV.N.R. 217

S y n t h e t i c S t u d i e s o f N e u r o t o x i n II f r o m V e n o m of C e n t r a l A s i a n C o b r a N a j a n a j a o x i a n a V.Deigin, V.Ulyashin, I.Mikhaleva,V.Ivanov

229

I n v e s t i g a t i o n on the S y n t h e s i s of S a l m o n C a l c i t o n i n II F r a g m e n t s G.Vlasov, V.Glushenkova, V.Lashkov, N.Kozhevnikova, L.Nadezhdina, L.Krasnikov, I.Ditkovskaja, O.Glinskaja,T.Komogorova

239

Total S y n t h e s i s of S o m a t o s t a t i n w i t h o u t H y d r o x y l G r o u p P r o t e c t i o n of H y d r o x y l A m i n o A c i d R e sidues Y.P.Shvachkin, S.K.Girin, A.P.Smirnova, A.A. Shishkina, N.M.Ermak

245

X Total Synthesis of Somatostatin-28 L.Moroder, M.Gemeiner, E.Jaeger, E.Wünsch

249

Synthesis, Structure and Membrane Properties of Gramicidin A Dimer Analogs L.Fonina, A.Demina, S.Sychev, A.Irkhin, V.Ivanov, J.Hlavacek

259

Cyclic Analogues of Bradykinin and Kallidin G.Chipens, F.Mutulis, N.Mishlyakova

269

Inhibition of RNA Polymerase by Analogs of Amaninamide T.Wieland, C.Birr, A.E.Vaisius, G.Zanotti

275

Structure-Activity Relationship of ActinBinding Peptides H.Faulstich

279

Synthesis, Spectral and Biological Properties of DSIP and its Analogs I.Mikhaleva, A.Sargsyan, T.Balashova, V.Ivanov

28 9

Polyoxyethyleneoxide(POE) Peptides, Models for Rhodopsin and Myoglobin E.Bayer

299

Chemical Mutation: Replacement of Reactive Site Residue P^=Lys in Bovine Kunitz Inhibitor by other Amino Acids and Concomitant Specificity Change H.Tschesche, H.R.Wenzel

301

Semisynthetic Modification of the N-Terminus of the Insulin A-Chain P.Trindler, D.Brandenburg

307

Enzymatic-Chemical Transformation of Porcine Insulin into Human Insulin E.N.Voluyskaja, S.P.Krasnoschokova, V.V.Knjazeva, M.N.Rjabtsev, S.M.Funtova, T.I.Zujanova, A.I.Ivanova, V.P.Fedotov, Yu.P.Shvachkin

315

Trypsin Catalyzed Peptide Synthesis: Modification of the ß-Chain C-Terminal Region of Insulin H.-G.Gattner, W.Danho, R.Knorr, H.Zahn

319

XI

Semisynthesis with Insulin-B-Chain E.E.Büllesbach, E.W.Schmitt, H.-G.Gattner, V.K.Naithani, J.Föhles

327

Investigation of the Influence of A1, B29 Amino Group Modification on the Structure and Biological Activity of Insulin G.Vlasov, N.Izvarina, N.Illarionova

337

Bacterial Cell Wall Glycopeptides: Synthesis, Conformation and Antitumor Activity T.Andronova, L.Rostovtseva, I.Sorokina, V.Mal'kova, V.Ivanov 343 Use of Dipeptide Substrates in Studies of Specific Features of Thrombin Catalysis V.K.Kibirev, S.B.Serebryany

353

Studies on the Efficacy of Preparative and Analytical HPLC in Peptide Chemistry W.Göhring, M.Gemeiner, L.Moroder, R.Nyfeler, E.Wünsch

359

A Method for Amino Acid Separation with a Microcolumn Chromatograph E.Ya.Kreindlin, V.S.Onoprienko, O.V.Evseeva, L.B.Kaminir

367

An Express Method for Determination of Absolute Configuration and Quantitity of Amino Acid Enantiomers in Mixtures N.A.Voskova, V.V.Romanov, G.A.Korshunova, Yu. P.Shvachkin

3 73

III. Structural Features of Proteins Structure-Activity Studies on Neurotoxin 1 from Sea Anemone Radianthus Macrodactylus E.Kozlovskaja, H.Vozhova, G.Elyakov

379

Cobra Venom Neurotoxins: Conformation and Interaction with the Acetylcholine Receptor V.Tsetlin, E.Karlsson, Y.Utkin, A.Arseniev, K.Pluzhnikov, A.Surin, V.Pashkov, V.Bystrov, V.Ivanov

389

XII

The Study of the Scorpion Neurotoxin Membrane Receptors N.Soldatov, V.Kovalenko, E.Grishin

399

Dehydration of Ionogenic Groups in Peptide Ligands on Receptor Surface G.V.Nikiforovich, S.A.Rozenblit, G.Chipens

407

The Study of the Structural and Functional Organization of the Somatotropin Molecule Y.A.Pankov, A.A.Bulatov, Y.M.Keda, T.A. Osipova, V.I.Pozdnyakov

415

Structural Features of Bacillus thuringiensis Crystal Protein V.M.Stepanov, G.G.Chestukhina, I.A.Zalunin, L.I.Kostina, A.L.Mikhailova

423

X-Ray Investigation of Three Dimensional Structure of Actinoxanthin V.Pletnev, A.Kuzin, S.Trakhanov, V.Popovich, I.Tsigannik

4 29

Aldimine Bond Migration in the Photochemical Cycle of Bacteriorhodopsin N.Abdulaev, V.Tsetlin, A.Kiselev, V.Zakis, Y.Ovchinnikov

435

Chemical Modofication of Purple Membrane Topography of Bacteriorhodopsin H.-D.Lemke, J.Bergmeyer, D.Oesterhelt

441

Formation and Unusual Properties of Bacterioopsin-Ag + -4-Dimethylaminochalcone Triple Complex A.Aldashev, A.Rodionov, E.Efremov, A.Shkrob 4 51 Peptide from Beef Heart Mitochondria Inducing Ion-Selevtive Channels on Planar Bilayer Membrane L.A.Pronevich, G.P.Mironov, G.D.Mironova 457 Functional Role of the Protein Component of the Ca -Transporting Glycoprotein from Beef Heart Homogenate and Mitochondria T.V.Sirota, L.A.Pronevich, G.D.Mironova

463

Optical Spectroscopy Study of Substrate Binding by Leucine Specific and Leucine - Isoleucine - Valine Binding Proteins from E.coli I.Nabiev, S.Trakhanov, A.Surin, T.Vorotyntseva, E.Efremov, V.Pletnev

4 67

Investigations of Immunoglobulin Structure in Solution V.Zav'yalov, V.Abramov, O.Loseva, V.Tishchenko E.Dudich, I.Dudich Conformational Lability of Immunoglobulin M Molecule E.Kaverzneva, F.Shamkova, Y.Khurgin, R.Kayushi na Structure-Functional Studies of DNA-Dependent RNA Polymerase from E.coli O.Chertov, V.Efimov, O.Chakhmakhcheva, Y.Smirnov, S.Tsarev, N.Skiba, T.Marchenko, V.Lipkin, E.Sverdlov Structure of the Proteolipid Subunit of the ATP Synthase J.Hoppe, W.Sebald Quaternary Structure and Reconstitution of Acetylcholine Receptor from Torpedo californica W.Schiebler, L.Lauffer, G.Bandini, F.Hucho

I

Isolation and Structure Elucidation of Peptides and Proteins

THE PRIMARY STRUCTURE OF DNA-DEPENDENT RNA POLYMERASE FROM E. ooli. NUCLEOTIDE SEQUENCE

OF rpoB GENE AND AMINO ACID

SEQUENCE OF THE (3-SUBUNIT

Valéry Lipkin, Galina Monastyrskaya, Valentin Gubanov, Sergey Guryev, Oleg Chertov, Vladimir Grinkevich, Irina Makarova, Tatjana Marchenko, Irina Polovnikova, Eugene Sverdlov, Yuri Ovchinnikov Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR

Introduction Elucidation of the transcription mechanism requires detailed knowledge of the active center's organization of RNA polymerase at the various stages of the RNA synthesis. This, in turn, can be obtained only after determining the primary and spatial structure of the enzyme. Earlier we had established the amino acid sequence of the a-subunit of E. coli DNA-dependent RNA polymerase resorting solely to the ordinary methods of protein chemistry

(1). In

the case of the g-and B'-subunits with their much higher molecular weights

(-155.000 and -165.000, respectively), such an

approach could no longer suffice.

Results One of the approaches to the structure determination of large protein molecules is their initial cleavage into a small number of fragments, which then can be analyzed by conventional methods. The search for the conditions of limited proteolysis of the g- and g'-subunits was undertaken. Considerable obstacles were encountered in the course of these studies owing

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter &. Co., Berlin • N e w York

4 to the fact that the RNA polymerase subunits are not the native proteins. However, the conditions for limited tryptic proteolysis of the B-subunit were found. These are an enzvme/substrate ratio of 1:500, temperature 0°C, 4 hr (2). Herein there seems, what we believe,to be an optimal set of large fragments (mol. wt.62.000, 52.000, 37.000, 24.000 and 10.000). Initial separation of the resultant hydrolysate was carrisd out by chromatography on Sephadex G—100 in 6 M guanidine hydrochloride. This yielded 10 fractions. Their analysis by polyacrylamide gel electrophoresis showed that all large fragments mentioned above are in the first three fractions, while the rest contain about 9 5 smaller peptides. 53 low molecular weight peptides were isolated from the hydrolysate. They consist of approximately 400 amino acid residues. Their sequencing was very useful for a further structure investigation (3). Isolation of the high molecular peptides proved difficult because of both the little hydrolytic specificity and the low yield of most products. So we could not use limited proteolysis as the main procedure for fS sequencing. The progress in DNA sequencing methods allowed to realize the possibility of using the genetic code to obtain information on the primary protein structure from the nucleotide sequences. However .here there are many pitfalls in the way, requiring considerable caution to avoid possible sources of error. In the first place the mRNA can undergo processing, leading to erroneous deduction of the protein structure. This holds particularly for eukaryotic cells, wherein "splicing" has been noted. Secondly, the protein itself can be processed. Thirdly, it is often difficult to recognize in the overall DNA structure the beginning of a structural gene. Moreover, one has to bear in mind that a single error (deletion or insertion) in the DNA sequence could lead to a completely erroneous amino acid sequence of the protein. Thus, primary structure determination of DNA cannot serve as a substitute for the direct sequencing of the protein. In view of this, we decided to utilize the methods of both protein

5 rpoC

EcoRI

rpo B

t ^D

Sail

E

—i

T

Fig. 1. EcoRI and Sail restriction cleavage m a p of the E.aoli DNA region including the structural genes (rpoB and rpoC) of the g-and g'- RNA polymerase subunits. and nucleotide chemistries, performing of the structural genes rpoB

the parallel

(g-subunit) and rpoC

sequencing

(3'-subunit)

and of the corresponding proteins. Knowledge of the nucleotide sequence of the pertinent DNA segments would permit

aligning

of the peptide fragments from the protein analysis into an uninterrupted polypeptide chain. Such an approach provides key to the most complicated problem in the primary analysis of high molecular

the

structure

proteins.

In Fig. 1 restriction endonucleases cleavage m a p of E. ool-i DNA region containing the structural genes of the g-and g '- subunits of the RNA polymerase given. We determined EcoRI-F

(rpoB and rpoC correspondingly)

the total sequence of the EcoRI-C

is

(4),

(5) and EcoRI-A - Hindlll fragments and partial

sequ-

ence of the EcoRI-G fragment carrying the beginning of the rpoB gene

(6). These fragments were obtained from DNA of

47 and rpoBC operon

18 transducing phages, containing the E. (7, 8), or corresponding plasmids by EcoRI

aoli

restric-

tion endonuclease digestion. In the case of EcoRI-A - Hindlll fragment EcoRI and Hindlll

digestions were used.

The fragments w e r e consecutivelv digested with one of the restriction endonucleases

(Sau 3AI, Hinf I, Hpa II and Taq I)

cleaving the DNA into relatively

small blocks. The resulting 32 subfragments were phosphorylated by means of |y- P|-ATP and

phage T4 polynucleotide kinase and the mixture was

separated

by electrophoresis on polyacrylamide gel. As a rule both

6

complementary chains obtained after denaturation of each subfragment and separation were analyzed. Their sequencing was performed by a modified Maxam-Gilbert procedure. As another method of the B-subunit polypeptide chain splitting, the digestion with Staphylococcus

aureus protease was chosen.

Initial fragmentation of the hydrolysate was performed by gel filtration on biogel P-4. This yielded four fractions. Subsequent separation of the peptides was achieved by chromatography on the cation exchanger AG-50Wx4 and paper chromatography. Fraction I contained a mixture of the largest peptides. To facilitate the separation and analysis of the peptides in this fraction the mixture was additionally digested with chymotrypsin. Altogether 3 peptides were isolated from fraction IV, 73 from fraction III, 48 frcm fraction II and 60 frcm frac tion I. In order to obtain the missing fragments, the exhaustive tryptic digestion of the B-subunit was carried out after modification of the lysine residues with citraconic anhydride. The tryptic peptides were separated according to the same scheme as that used for staphylococcal peptides. After removal of the citraconic protection the high molecular weight peptides were subjected to additional tryptic cleavage at the lysine residues. In order to determine the primary structure of the B'-subunit its cyanogen bromide cleavage was carried out. For separation of the resulting peptides we used gel filtration, paper and thin-layer chromatographies, electrophoresis in acetate cellulose slabs and butanol exstraction of hydrophobic peptides. The tryptic

digestion of the B'-subunit was also carried out af-

ter modification of the lysine residues with citraconic anhydride. The amino acid sequence of the peptide fragments determined up to now covers more than 80% of the total B-subunit polypeptide chain and about 30% of the B 1 -subunit polypeptide chain. During this investigation the comparison of the nucleotide and amino acid sequence was carried out continually. The search for similarities between the amino acid sequences of the

7 TTC CGG TCA ACA AAA TAG TGT TGC ACA AAC TGT CCG CTC AAT GGA CAG ATG GGT CGA CTT GTC AGC GAG CTG AGG AAC CCT ATG GTT TAC TCC TAT ACC GAG AAA AAA CGT ATT CGT AAG GAT TTT GGT AAA CGT CCA CAA GTT CTG GAT GTA CCT TAT CTC Het-Va1-Tyr-Ser-Tyr-Thr-Glu-lys-lys-Arg-l I e-Ar9-Lys-Asp-Phe-6 » y-Lys -Arg»?ro-ttIn-V«>-leu-Atp-Vat-Pro-Ty r -leu« CTT TCT ATC CAG CTT GAC TCG TTT CAG AAA TTT ATC GAG CAA GAT CCT GAA GGG CAG TAT GGT CTG GAA GCT GCT TTC CGT Leu-Ser-lle-Gln-Leu-Asp-Ser-Phe-Gln-Lys-Phe-lle-6lu-61n»Asp»Pro«Glu-Gly-Glri»Tyr-G1yleu»Clu-Ala-Al»*Phe*ArflTCC GTA TTC CCG ATT CAG AGC TAC AGC GGT AAT TCC GAG CTG CAA TAC GTC AGC TAC CGC CTT GGC GAA CCG GTG TTT GAC S e r - V a l - P h e - P r o - I I e - G 1 r > - S e r - T y r - S e r - G 1 y -Asn- S e r - G I u - L e u - G 1 n - T y r - V a 1 - S e r - T y r » A r g - l e u - G t y « G t u - P r o - V » ' - P h « - A » p « GTC Í A G GAA TGT CAA ATC CGT GGC GTG ACC TAT TCC GCA CCG CTG CGC GTT AAA CTG CGT CTG GTG ATC TAT GAG CGC GAA Val-Gln»Glu»Cy&-Gln-Ile-Arg-Gly-VaI»Thr-Tyr-Ser-Ala-Pro-leu-Arg-Val-Lys-leo-Arg-leu-Val-I Ie-Tyr»6tu-Arg»61 w GCG CCG GAA GGC ACC GTA AAA GAC ATT AAA GAA CAA GAA GTC TAC ATG GGC GAA ATT CCG CTC ATG ACA GAC AAC GGT ACC Al a-Pro-Çlu-Gl y-Thr-Va 1 - f c y s - A s p - M e - l y s - G l u - G l n - G 1 u - V a l - T y r » H e i - G 1 y - G l u - H e - P r o - l e u « H e t - T h r - A s p « A s r i - G » y - T h r TTT GTT ATC AAC GGT ACT GAG CGT GTT ATC GTT TCC CAG CTG CAC CGT AGT CCG GGC GTC TTC TTT GAC TCC GAC AAA GGT P h e - V a I - I l e - A s n - G l y - T h r - G l u - A r g - V a l - l I e - V a ) - S e r - G I n - l e u - H i s - A r g - S e r - P r o - G I y•Va I - P h e - P h e - A s p - S e r - A » p - l y S - G 1 y AAA ACC CAC TCT TCG GGT AAA GTG CTG TAT AAC GCG CGT ATC ATC CCT TAC CGT GGT TCÊ TGG CTG GAC TTC GAA TTC GAT l y s - T h r » H i s - S e r - S e r - G l y - L y s - V a I - L e u - T y r - A s n - A l a - A r g - I l e - l ) e - P r o - T y r - A r g - G 1 y • S e r - T r p - l e u - A » p - P h e - G 1u-Phe-AspCCG AAG GAC AAC CTG TTC GTA CGT ATC GAC CGT CGC CGT AAA CTG CCT GCG ACC ATC ATT CTG CGC GCC CTG AAC TAC ACC Pro-lys-A>p-Asn-leu-Phe-Vat-Arg-l1e-Asp-Arg-Arg«Arg-Lys-leu-Pro-Ala-Th r - I l e - l le-leu-Arg-Ala-leu-A>n-Tyr-ThrACA GAG CAG ATC CTC GAC CTG TTC TTT GAA AAA GTT ATC TTT GAA ATC CGT GAT AAC AAG CTG CAG ATG GAA CTG GTG CCG Th r - G Iu-G I n » I I e - 1 e u - A s p - l e u - P h e - P h e - G 1u-Ly s-Va I - I l e - P h e - G l u - l I e » A r g - A s p - A s n - L y * - L e u - G 1 n - H e t - G 1 u - l e u - V » I - P r o GAA CGC CTG CGT GGT GAA ACC GCA TCT TTT GAC ATC GAA GCT AAC GGT AAA GTG TAC GTA GAA AAA GGC CGC CGT ATC ACT G)u-Arg-Leu-Arg-G1y-G)u-Thr-Ala-Ser-Phe-Asp-ne-Glu-Ala-Asn-C1y-Lys-Val-Tyr»Val-Glu-lys-G1y-Arg-Arg-lle-ThrGCG CGC CAC ATT CGC CAG CTG GAA AAA GAC GAC CTC AAA CTG ATC GAA GTC CCG GTT GAG TAC ATC GCA GGT AAA GTG GTT A l a - A r g « H i s - I l e - A r g - G I n - L e u - G Iu-Ly s - A s p - A s p - V a l - l y s - l e u - l l e - C l u - V a l - P r o - V a l - G l u - T y r - M e - A > e - 6 » y - l y S - V a i - V a 1 GCT AAA GAC TAT ATT GAT GAG TCT ACC GGC GAG CTG ATC TGC GCA GCG AAC ATG GAG CTG AGC ¿ T G GAT CTG CTG GCT AAG Ala-Ly>»Asp-Tyr-Ite-Asp-G1u-Ser-Thr-Gly-Glu-Leu-Me-Cys-Ala-Ala-Asn-Het-Glu-leu-Ser-Leu-Asp-Leu-leu-Ala-Lys» CTG AGC CAG TCT GGT CAC AAG CGT ATC GAA ACG CTG TTC ACC AAC GAT CTG GAT CAC GGC CCA TAT ATC TCT GAA ACC TTA Leu-Ser-Gln-Ser-Gly-Mis-Lys-Arg-Ile-Clu-Thr-Leu-Phe-Thr-Asn-Asp-Leu-Asp-His-Gly-Pro-Tyr-Me-Ser-Glu-Thr-LeuCGT GTC GAC CCA ACT AAC GAC CGT CTG AGC GCA CTG GTA GAA ATC TAC CGC ATG ATG CGC CET GGC GAG CCG CCG ACT CGT Arg-Val-Asp-Pro-Thr-Asn-Asp-Arg-Leu-Ser-Ala-Leo-Val-GIu-lle-Tyr-Arg-Wet-HefArg-Pro-Gly-Glu-Pro-Pro»Thr»Arg»

AAC CGT TCT CTG CTG CGC GAA GAA ATC GAA GGT TCC GGT ATC CTG AGC AAA GAC GAC ATC ATT GAT GTT ATG AAA AAG CTC A s n - A r g - S e r - l e u - l e u - A r g - G I u-GI u - I le-61 u - G l y - S e r - G l y - I l e - l e u - S e r - l y s - A > p - A i p - M e - M e - A s p - V » l - H e t - l y i - l y » - l c w ATC GAT ATC CGT AAC GGT AAA GGC GAA GTC GAT GAT ATC GAC CAC CTC GGC AAC CGT CGT ATC CGT TCC GTT GGC GAA ATG I le-Asp-t le-Arg-Asn-G I y - l y s - G l y - G l u - V a l - A s p - A s p - I l e - A s p - H i s - l e u - G 1 y - A > n - A r g - A r g - l l e - A r g - S e r - V a l - G l y - G l u - H e t GCG GAA AAC CAG TTC CGC GTT GGC ÈTG GTA CGT GTA GAG CGT GCG GTG AAA GAG CGT CTG TCT CTG GGC GAT CTG GAT ACC A l a - G l u-As n» G 1 n - P h e - A r g - V a I - G ) y - l c u - V a l - A r g - V a 1 - G l u - A r g - A l a - V a I - L y s - G l u - A r g - L e u - S e r - l e u - G I y - A s p - L e u * A s p - T h r CTG ATG CCA CAG GAT ATG ATC AAC GCC AAG CCG ATT TCC GCA GCA GTG AAA GAG TTC TTC GGT TCC AGC CAG CTG TCT CAG Leu-He t - P r o - G l n - A s p - H e t - l l e - A s n - A l a - l y s - P r o - l l e - S e r - A l a - A l a » V a l » l y s - G ) u - P h e « P h e - G I y - S e r - S e r - G l n - l e u - S e r - G I n TTT ATG GTC CAG AAC AAC CCG CTG TCT GAG ATT ACG CAC AAA CGT CGT ATC TCC GCA CTC GGC C Í A GGC GGT CTG ACC CGT Phe-Het-Val-GIn-Asn-Asn-Pro-leu-Ser-Glu-lle-Thr-Hi s-Lys-Arg-Arg-lle-Ser-Ala-leu-Gly-Pro-Gly-Gly-leu-Thr-ArgGAA CGT GCA GGC TTC GAA GTT CGA GAC GTA CAC CCG ACT CAC TAC GGT CGC GTA TGT CCA ATC GAA ACC CCT GAA GGT CCG Glu-Arg-Ala»61y-Phe-Glu-Val-Arg-Asp-Val-His-Pro»Thr-His-Tyr»Gly*Arg-Val-Cys*Pro-lle-Glu»Thr-Pro-Glu»Gty-Pro» AAC ATC GGT CTG ATC AAC TCT CTG TCC GTG TAC GCA CAG ACT AAC GAA TAC GGC TTC CTT GAG ACT CCG TAT CGT AAA GTG A s n - l l e - G l y - L e u - l l e - A s n - S e r - l e u - S e r - V a l - T y r - A I a•G I n - T h r - A s n - G I u - T y r - G 1 y - P h e - I e u - G I u - T h r - P r o - T y r - A r g - I y > - V a I ACC GAC GGT GTT GTA ACT GAC GAA ATT CAC TAC CTG TCT GCT ATC GAA GAA GGC AAC TAC GTT ATC GCC &AG GCG AAC TCC Thr-Asp-Gly-Val-Val-Thr»Asp-Glu-lle-His-Tyr-Leu-Ser-Ala-lle-Glu-Glu-Gly-Asn-Tyr-Val»lle»Ala-Gln»AI«-Asn-SefAAC TTG GAT GAA GAA GGC CAC TTC GTA GAA GAC CTG GTA ACT TGC CGT AGC AAA GGC GAA TCC AGC TTG TTC AGC CGC GAC Asn-leu-Asp-Glu-Glu-GI y H i s-Phe-Vat-6lu-Asp-leu-Va1-Thr-Cys-Arg»Ser«Lys-Cly-61 u-Ser-Ser-lcu-Phe-Ser-Arg-A»pCAG GTT GAC TAC ATG GAC GTA TCC ACC CAG CAG GTG GTA TCC GTC GGT GCG TCC CTG ATC CCG TTC ¿ T G GAA CAC GAT GAC Gln-Val-Asp'Tyr-Het-Asp-Val-Ser-Thr-Gln-Gln-Val-Val-Ser-Val-Cly-Ala-Ser-leu-l)e-Pro-Phe-leu-Glu-HI$-A»p-A»pGCC AAC CGT GCA TTG ATG GGT GCG AAC ATG CAA CGT CAG GCC GTT CCG ACT CTG CGC GCT GAT AAG CCG CTG GTT GGT ACT Ala-Asn-Arg-Ala-leu-het-Gly-Ala-Asn-Het-Gln-Arg-Gln-Ala-VaI-Pro»Thr-L - G t y - V I - S e f G I y-Th r - V « I - I ¡ • • A i p - V i l - G l n - V a l - P h e - T h r - A r q - A i p - G l y - V a l - G l u - L y t - A s p - L y s - A r g - A l a CTG GAA ATC GAA GAA ATG CAG CTC AAA CAO GÇG AAG AAA GAC CTG TCT GAA GAA CTG CAG ATC CTC GAA GCG GGT CTG TTC AGC CGT ATC CGT GCT GTG CTG GTA GCC GGT GGC GTT GAA GCT GAG AAG CTC GAC AAA CTG CCG CGC GAT CGC TGG CTG GAG S e r-A rq- I ( e - A r q - A 1 a - V a I - l « w - V a I - A I a - G ) y - G I y - V » l - G l u - A l a - G l u - L y » - l e M - A « p - l y » - L e u - P r o - A r g - A » p - A r g - T r p - L e u - G l u CTG GGC CTG ACA GAC GAA GAG AAA CAA AAT CAG CTG GAA CAG CTG GCT GAG CAG TAT GAC GAA CTG AAA CAC GAG TTC GAG Leu-Gly-L«u-Thr-A»p-Glu-GU-Ly»-G(n-A»n'G)n-L«u-Glu-Gln-Leu-Ala-Glu-Gln-Tyr-Atp-Glu-Leu-Lys-His-Glu-Phc-G)uAAG AAA CTC GAA GCG AAA CGC CGC AAA ATC ACC CAG GGC GAC GAT CTG GCA CCG GGC GTG CTG AAG ATT GTT AAG GTA TAT L y s - l y » - L e u - G l u - A l a - L y » - A r g - A r g - L y » - t I e - T h r - G In-G 1 y - A i p - A » p - L e u - A I a - P r o - G 1 y-Va 1 - L e u - L y s - I l e - V a l - L y s - V a l -Ty r CTG GCG GTT AAA CGC CGT ATC CAG C Î T GGT GAC AAG ATG GCA GGT CGT CAC GGT AAC AAG GGT GTA ATT TCT AAG ATC AAC Leu-Ala-Val-Lyt-Arg-Arg-lle-6tn-Pro-Gly-A»p-ty»-Het-Ala-Gly-Arg-His-Gly-Asn-tyi-Gly-Val-lle-Ser-Ly»-lte-A»nCCG ATC GAA GAT ATG CCT TAC GAT GAA AAC GGT ACG CCG GTA GAC ATC GTA CTG AAC CCG CTG GGC GTA CCG TCT CGT ATG P r o - I l e - G l u - A » p - H e t - P r o - T y r - A t p - G l u - A s n - G l y - T h r - P r o - V a l - A » p - l I e -Va I - L e u - A s n - P r o - L e u - G 1 y - V a 1 - P r o - S e f - A r q - H e t -

CAG CAA GAA GTC GCG AAA CTG CGC GAA TTC ATC CAG CGT GCG TAC GAT CTG GGC GCT GAC GTT CGT CAG AAA GTT GAC CTG - A I « - L y s L e u A r q G l u -Phe 1 e G i n A r q A l a T y r Asp L e u G l y A l a Asp V a l A r q Asp L e u « Ly. AGT ACC TTC AGC GAT GAA GAA GTT ATG CGT CTG GCT GAA AAC CTG CGC AAA GGT ATG CCA ATC GCA ACG CCG GTG TTC GAC - S e r -Asp - G l u G l u V a l Het -Arq L e u A l a G u Asn L e u A r q L y s u i y He t P r o 1 l e A l a T h r P r o Va 1 AspGGT GCG AAA GAA GCA GAA ATT AAA GAG CTG CTG AAA CTT GGC GAC CTG CCG ACT TCC GGT CAG ATC CGC CTG TAC GAT GGT G l y - A l a •Ly» - G l u - A l a - G l u l l e L y , G l u L e u L e u L y s L e u G y Asp L e u P r o Thr S e r G l y G i n 1 e A r g L e u l y r Asp G l y CGC ACT GGT GAA CAG TTC GAG CGT CCG GTA ACC GTT GGT TAC ATG TAC ATG CTG AAA CTG AAC CAC CTG GTC GAC GAC AAG A r g •Thr - l i l y - G l u G i n - P h e G u * r q P r o Va 1 Thr V a t G l y T y r Met l y r Het L e u L y s L e u A s n HI s L e u Va 1 A s p A s p L y s ATG CAC GCG CGT TCC ACC GGT TCT TAC AGC CTG GTT ACT CAG CAG CCG CTG GGT GGT AAG GCA CAG TTC GGT GGT CAG CGT H e t •Hi S -Arq Thr G l y S e r Ty r S e r L e u Va 1 Thr G i n G i n P r o L e u G l y G l y L y s A l a G i n P h e G l y G l y G i n A r r TTC G6G GAG ATG GAA GTG TGG GCCLCTG GAA GCA TAC GGC RCA GCA TAC ACC CTG CAG GAA ATG CTC ACC GTT AAG TCT GAT Gly ••Het G l u Va 1 U p A l a L e u G l u A l a l y r G l y A l a A l a T y r Thr L e u G i n G l u He t L e u T h r Va 1 L y s S e r AspGAC GTG AAC GGT CGT ACC AAG ATG TAT AAA AAC ATC GTG GAC GGC AAC CAT CAG ATG GAG CCG GGC ATG CCA GAA TCC TTC Ar q Thr L y s Met T y r L y » Asn 1 e Va l Asp G y Asn H s G i n He t Asp G y H e t P r o G l u S e r PheAAC GTA TTG TTG AAA GAG ATT CGT TCG CTG GGT ATC AAC ATC GAA CTG GAA GAC GAG TAA TTC TCG CTC AAA CAG GTC A Asn Lew L e u L y s G l u 1 l e A r q S e r L e u G l y Asn G l u L e u G l u Asp G l u Tt R CTG CTG TCG GGT TAA AAC CCG GCA GCG GAT TGT GCT AAC TCC GAC GGG AGC AAA TCC GTG AAA GAT TTA TTA AAG TTT CTG Het L y s Asp L e u L e u L y s P h e l e u AAA GCG CAG ACT AAA AC f. GAA GAG TTT GAT GCG ATC AAA ATT GCT CTG GCT TCG CCA GAC ATG ATC CGT TCA TGG TCT TTC Lys G l u Phe Asp A l a 1 l e L y s 1 e A l a L e u Ser Asp H e t Arg S e r I r p Ser Phe* lys GGT GAA 6TT AAA AAG CCG GAA ACC ATC AAC TAC CGT ACG TTC AAA CCA GAA CGT GAC GGC CTT TTC TGC GCC CGT ATC TTT Gly Leu L y * L y s P r o G u Thr 1 l e Asn l y r A r g Thr Phe L y s P r o G l u A r g Asp C y s A l a A r g 1 e PheGGG CCG GTA AAA GAT TAC GAG TGC CTG TGC GGT AAG TAC AAG CGC CTG AAA CAC CGT GGC GTC ATC TGT GAG AAG TGC GGC Gly G l u L y s Cys G l y L y » Asp l y r G l u Cys L e u Cys G l y L y s T y r L y s A r g L e u L y s H i s A r g GTT GAA GTG ACC CAG ACT AAA GTA CGC CGT GAG CGT ATG GGC CAC ATC GAA CTG GCT TCC CCG ACT GCG CAC ATC TGG TTC Va 1 G n Thr L y s V a l A r g A r g G l u A r g Het G l y H I S 1 1 G l u L e u A l a H I S I l e I r p PheCTG AAA TCG CTG CCG TCC CGT ATC GGT CTG CTG CTC GAT ATG CCG CTG CGC GAT ATC GAA CGC GTA CTG TAC TTT GAA TCC Arg lyr LysGlu SerLewS e r - Arg- l l e G l y L e u L e u L e u Asp He c P r o L e u A r q Asp 1 l e TAT GTG GTT ATC GAA GGC GGT ATG ACC AAC CTG GAA CGT CAG CAG ATC CTG ACT GAA GAG CAG TAT CTG GAC GCG CTG GAA Tyr-Val-Val-lle-Glu-Gly-Gly-Met-Thr-Aïn-Leu-Glu-Arg-Gln-Gln-lle-Leu-Thr-Gtu-Glu-G1n-Tyr-Leu-A»p-Ala-Leu-Glu-

Fig. 2. The nucleotide sequence of the rpoBC segment, the total amino acid sequence of the g-subunit and the N-terminal amino acid sequence of the 6'-subunit of E. ooli RNA polymerase. The restriction EcoRI cleavage sites dividing the fragments EcoRI-G, EcoRI-C and EcoRI-F are situated between nucleotides 640-641 and 3508-3509. Here and in Fig. 3 the nucleotide sequence of the complementary DNA chain, equal to the sequence of mRNA, is given. The underlined amino acid sequences are those the structure of which has been determined from analysis of corresponding peptides. C* refer to 5-methylcytidine residues.

9 1-75 1-25

GAA TTC GGT CGT ACT AAA GAA AGC TAC AAA GTA CCT TAC GOT GCG GTA CTG GIG AAA GGC GAT GGC GAA CAG GTT G ^ u - P h e - G l y - A ^ g - ^ - l J ' 2 - G ' u - S e r " T y r " l - y 5 " V a ' " p ^ 0 " 1 ^ r " G , y " A , " " V a , -Leu-Ala-Lys-Gly-Asp-Gly-Glu-Gln-Val -

76-150 26-50

GCT GGC GGC GAA ACC GTT GCA AAC TGG GAC CCG CAC ACC ATG CCG GTT ATC ACC GAA GTA AGC GGT TTT GTA CGC A l a - G l y - G l y - G l u - T h r - V a I - A l a - A s n - l r p - A s p - P r o - H i s-Thr-Met-Pro-Val-11e-Thr-G1u-Va1-Ser-GTy-Phe-Val-Arq-

151-225 51-75

TTT ACT GAC ATG ATC GAC GGC CAG ACC ATT ACG CGT CAG ACC GAC GAA CTG ACC GGT CTG TCT TCG CTG GTG GTT Phe-Thr-Asp-Met-I1e-Asp-G1y-G1n-Thr-Ile-Thr-Arg-Gln-Thr-Asp-G1u-Leu-Thr-Gly-Leu-Ser-Ser-Leij-Val-Va1-

226-30C 76-100

CTG GAT TCC GCA GAA CGT ACC GCA GGT GGT AAA GAT CTG CGT CCG GCA CTG AAA ATC GTÏ GAT GCT CAG GGT AAC Leu-Asp-5er-Ala-Glu-Arg-Thr-Ala-G^-Gly-Lys-Asp-Leu-Arg-Pro-Ala-Leu-Lys-Ile-Val-Asp-Ala-Oln-Gly-Asn-

301-375 101-125

GAC GTT CTG ATC CCA GGT ACC GAT ATG CCA GCG CAG TAC TTC CTG CCG GGT AAA GCG AIT GTT CAG CTG GAA GAT Asp-Val-Leu-Ile-Pro-Gly-Thr-Asp-Mrt-Pro-Ala-Gln-Tyr-Phe-Leu-Pro-Gly-Lys-Ala-Me-Val-Gln-Leu-Glu-Asp-

376-450 126-150

C.GC GTA CAG ATC AGC TCT GGT GAC ACC CTG GCG CGT ATT CCG CAG GAA TCC GGC GGT ACC AAG GAC ATC ACC GGT Gly-Val-Gln-lle-Scr-Ser-Gly-Asp-Thr-Leu-Ala-Arg-lle-Pro-Gln-Glu-Ser-Gly-Gly-Thr-Lys-Asp-Ile-Thr-Gly-

451-525 151-175

GGT CTG CCG CGC GTT GCG GAC CTG TTC GAA GCA CGT CGT CCG AAA GAG CCG GCA ATC CTG GCT GAA ATC AGC GGT Gly-Leu-Pro-Arg-Val-Ala-Asp-leu-Phe-Glu-Ala-Arg-Arg-Pro-Lys-Glu-Pro-Ala-11e-Leu-A1a-Glu-IIe-5er-Gly-

526-600 176-200

ATC GTT TCC TTC GGT AAA GAA ACC AAA GGT AAA CGT CGT CTG GTT ATC ACC CCG GTA GAC GGT AGC GAT CCG TAC Ile-Val-Ser-Phe-Gly-Lys-Glu-Thr-Lys-Gly-Lys-Arg-Arg-Leu-Val-1le-Thr-Pro-Val-Asp-Gly-Ser-Asp-Pro-Tyr-

601-675 201-225

GAA GAG ATG ATT CCG AAA TGG CGT CAG CTC AAC GTG TTC GAA GGT GAA CGT GTA GAA CGT GGT GAC GTA ATT TCC Glu-Glu-Met-Ile-Pro-Lys-Trp-Arg-Gln-Leu-Asn-Val-Phe-Glu-Gly-Glu-Arg-Val-Glu-Arg-Gly-Asp-Val-Ile-Ser-

676-750 226-250

GAC GGT CCG GAA GCG CCG CAC GAC ATT CTG CGT CTG CGT GGT GTT CAT GCT GTT ACT CGT TAC ATC GTT AAC GAA Asp-Gly-Pro-Glu-Ala-Pro-Hls-Asp-llc-Leu-Arg-Leu-Arg-Gly-Val-His-Ala-Val-Thr-Arg-Tyr-I le-Val-Asn-Glu-

751-825 251-275

GTA CAG GAC GTA TAC CGT CTG CAG GGC GTT AAG ATT AAC GAT AAA CAC ATC GAA GTT ATC GTT CGT CAG ATG CTG Val -Gin-Asp-Val-Tyr-Arg-Leu-Gln-Gly-Val-Lys-Ile-Asn-Asp-Lys-His-Ile-Glu-Val-1le-Val-Arg-Gln-Met-Leu-

826-9Û0 276-300

CGT AAA GCT ACC ATC GTT AAC GCG GGT AGC TCC GAC TTC CTG GAA GGC GAA CAG GTT GAA TAC TCT CGC GTC AAG Arg-Lys-Ala-Thr-Ile-Val-Asn-Ala-Gly-Ser-Ser-Asp-Phe-Leu-Glu-Gly-Glu-Gln-Val-Glu-Tyr-Ser-Arg-Val-Lys-

30J-975 301-325

ATC GCA AAC CGC GAA CTG GAA GCG AAC GGC AAA GTG GGT GCA ACT TAC TCC CGC GAT CTG CTG GGT ATC ACC AAA lle-Ala-Asn-Arg-Glu-l.eu-Glu-Ala-Asn-Gly-Lys-Val-Gly-Ala-Thr-Tyr-Ser-Arg-Asp-Leu-Leu-Gly-lle-Thr-Lys-

976-1050 326-350

GCG TCT CTG GCA ACC GAG TCC TTC ATC TCC GCG GCA TCG TTC CAG GAG ACC ACT CGC GTG CTG ACC GAA GCA GCC Ala-Ser-Leu-Ala-Thr-Glu-Ser-Phe-Ile-Ser-Ala-Ala-Ser-Phe-Gln-Glu-Thr-Thr-Arg-Val-Lcu-Thr-Glu-Ala-Ala-

I05I-1125 351-375

GTT GCG GGC AAA CGC GAC GAA CTG CGC GGC CTG AAA GAG AAC GTT ATC GTG GGT CGT CTG ATC CCG GCA GGT ACC Val-Ala-Gly-Lys-Arg-Asp-Glu-leu-Arg-Gly-Leu-Lys-Glu-Asn-Val-lle-Val-Gly-Arg-Leu-lle-Pro-Ala-Gly-Thr-

I126-1200 376-400

GGT TAC GCG TAC CAC CAG CAT CGT ATG CGT CGC CGT GCT GCG GGT GAA GCT CCG GCT GCA CCG CAG GTG ACT GCA Gly-Tyr-Ala-Tyr-His-Gln-Asp-Arq-Met-Arg-Arg-Arg-Ala-Ala-Gly-Glu-Ala-Pro-Ala-Ala-Pro-Gln-Val-Thr-Ala-

I20I-I277 401-421

GAA GAC GCA TCT GCC AGC CTG GCA GAA CTG CTG AAC GCA GGT CTG GGC GGT TCT GAT AAC GAG TAA TCGTTAATCCG CH'-Asp-Ala-Ser-Ala-Ser-Leu-Ala-Glu-Leu-Leu-Asn-Ala-Gly-Leu-Gly-Gly-Ser-Asp-Asn-Glu Ter

1278-1376

CAAATAACGT.AAAAACCCGC.TTCI;GCGGGTTTTT,TTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGAATTTCTGAATACTCATAA

1377-1475

TCAATGTAGAGATGACTAATATCCTGAAACTGACTGAACTAATTGAGTCAAACTCGGCAAGGATTCGATACTATTCCTGTGTAACTTTCTTAAGGAACG

¡476-1574

AGAATGAAACAGGAAGTGGAAAAGTGGCGACCTTTTGGACATCCGGATGGTGATATTCGTGATTTATCATTTCTTGATGCTCATCAGGCTGTCTACGTT

1575-1673

CAGCATCATGAGGGCAAAGAGCCTTTAGAGTATCGCTTTTGGGTTACCTACTCTCTTCACTGCTTCACAAAAGATTATGAACATCAGACGAACGAAGAA

1674-1772

AAACAATCGTTAATGTACCACGCGCCTAAAGAATCTCGTCCCTTCTGCCAGCACCGTTATAACTTAGCGCGCACACACTTAAAAAGAACTATTTTGGCG

1773-187I

CTGCCAGAAAGCAACGTTATTCATGCCGGGTATGGTAGCTATGCCGTGATTGAGGTGGACTTAGACGGAGGAGATAAGGCATTTTACTTTGTTGCGTTC

1072-1970

AGGGCTTTCAGGGAAAAGAAAAAACTCCGTTTGCATGTAACTAGCGCTTATCCCATTTCTGAAAAACAGAAAGGTAAATCAGTGAAATTTTTCACCATT

1971-2069

GCCTACAACTTATTGAGAAATAAGCAGCTTCCTCAGCCCTCAAAATAACAAAACCCACCTTAAGGTGGGTTTCGCCAGAGAATTATCTCTGGTATTCAG

2070-2168

AACGCCATTACCGGACTTTGCCTTGACCTTGCGATAATCGCAGGTTGCGGGATGTCTGAATTTCTTCAGTCTGCTGCATCCTGGAAGATGAGAACATGT

2169-2267 2268-2306

GTTCTTATTTTCGTCTCTATCATAGTTGAGTATTTACTCTCTTACAATCAGATCTCTTTCATTGCTCAACAGGCGATGGCTTCAGACTTTGCATTACGG AATTTTTAAGAAAGGCAGGGCGAAACGAGGAAGAAGCTT

Fig. 3. The nucleotide sequence of the EcoRI-A - Hindlll fragment and the C-terminal amino acid sequence of the e'-subunit of E. aoli RNA polymerase.

10

peptides and the nucleotide sequences of the corresponding DNA fragments was carried out by means of a computer. A conjunction of the methods of protein and nucleotide chemistry for the combined structural investigation of a protein and DNA sharply accelerated and considerably simplified the solution of both problems and enhanced the reliability of the structural analysis. As a result the nucleotide sequences of the two segments of the rpoBC operon (4714 and 2306 base pairs) embracing the entire rpoB gene, the initial and terminal parts of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the 3- subunit, comprising 1342 residues

(9, 10), and the N- and C-terminal sequences of the

g'-subunit (176 and 421 residues) were determined (Eig. 2, 3).

References 1. 2.

Ovchinnikov, Yu.A., Lipkin, V.M., Modyanov, N.N., Chertov, 0.Yu., Smirnov, Yu.V.: FEBS Letters 76, 108-111 (1977). Marchenko, T.V., Modyanov, N.N., Lipkin, V.M., Ovchinnikov, Yu.A.: Bioorg. Khim. 6, 325-331 (1980).

3.

Lipkin, V.M., Marchenko, T.V., Khokhryakov, V.S., Polovnikova, I.N., Potapenko, N.A., Modyanov, N.N., Ovchinnikov, Yu.A.: Bioorg. Khim. 6, 332-342 (1980).

4.

Ovchinnikov, Yu.A., Sverdlov, E.D., Lipkin, V.M., Monastyrskaya, G.S., Chertov, O.Yu., Gubanov, V.V., Guryev, S.O., Modyanov, N.N., Grinkevich, V.A., Makarova, I.A., Marchenko, T.V., Polovnikova, I.N.: Bioorg. Khim. 6, 655-665 (1980).

5.

Monastyrskaya, G.S., Gubanov, V.V., Guryev, S.O., Lipkin, V.M., Sverdlov, E.D.: Bioorg. Khim. 6, 1106-1109 (1980).

6.

Monastyrskaya, G.S., Gubanov, V.V., Guryev, S.O., Lipkin, V.M., Sverdlov, E.D.: Bioorg. Khim. 6, 1423-1426 (1980).

7.

Mindlin, S.S., Ilyina, T.S., Gorlenko, Ch.M., Hachikyan, N.A., Kovalev, Yu.N.: Genetika 12, 116-130 (1976).

8.

Kirschbaum, J.B., Konrad, B.E.: J. Bacteriology 116, 517526 (1973).

9.

Ovchinnikov, Yu.A., Monastyrskaya, G.S., Gubanov, V.V., Guryev, S.O., Chertov, O.Yu., Modyanov, N.N., Crinkevich, V.A., Makarova, I.A., Marchenko, T.V., Polovnikova, I.N., Lipkin, V.M., Sverdlov, E.D.: Dokl. Akad. Nauk SSSR 253,

11

994-999 (1980). 10.

Ovchinnikov, Yu.A., Monastyrskaya, G.S., Gubanov, V.V., Guryev, S.O., Chertov, O.Yu., Modyanov, N.N., Grinkevich, V.A., Makarova, I.A., Marchenko, T.V., Polovnikova, I.N., Lipkin, V.M., Sverdlov, E.D.: Eur. J. Biochem.116, 621-629 (1981) .

STUDY ON THE PRIMARY STRUCTURE OF THE ELONGATION FACTOR G FROM

E.ooli

Yuli B. Alakhov, Ludmila P. Motuz, Natalia V. Dovgas and Yuri A. Ovchinnikov Institute of Protein Research, Academy of Sciences of the USSR, 142292 Poustchino, Moscow Region, USSR

Introduction The bacterial protein-synthesizing system is known to contain three soluble proteins participating in synthesis of polypeptide chains on the ribosome at the elongation stage. One of them, the elongation factor G (EF-G), catalyzes the GTP-dependent translocation of the peptidyl-tRNA'mRNA complex from the A-site into the P-site on the ribosome. This is accompanied by the release of the deacylated tRNA from the P-site and the removal of the tRNA from the ribosome

(1).

EF-G represents a protein consisting of one polypeptide chain with M r of about 80,000. The use of traditional methods for the determination of the primary structure did not seem expedient for such a large protein. Therefore J choosing the strategy of structural analysis,we opted at the first stage for limited proteolysis which splits the protein molecule into a small number of large fragments relatively stable to further protease action. It has been shown earlier that mild tryptic hydrolysis splits the elongation factor G into five fragments (2-4), four of them

(T4-T7) [for the fragments nomenclature

see (3)] encompass the whole protein polypeptide chain. Thus, the study of the EF-G primary structure amounted to the determination of the structure of these four fragments and the search for the peptides which assemble the fragments of li-

C h e m i s t r y of P e p t i d e s a n d P r o t e i n s , V o l . 1 © 1982 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

14 mited trypsinolysis into one polypeptide chain. Results From the data of amino acid analysis and determination of the molecular mass of peptide fragments which are the products of tryptic hydrolysis it can be concluded that limited trypsinolysis does not result in splitting off of any notable regions of the polypeptide chain (3). At the same time, elucidation of the structure of peptides obtained during the study of the EF-G cyanogen bromide

cleavage

permitted us to assemble all

the fragments of limited trypsinolysis into one polypeptide chain. Hence it follows that there are really no insertions between the fragments of limited trypsinolysis, i.e. trypsin produces point disruptions of the EF-G polypeptide chain (4). Fragment Tg is N-terminal, it is followed along the chain by fragment T^ which contains the functionally important cysteine residue. The GTP-binding site is located within this fragment (5). Fragment T^ is followed by fragment T^ and, finally, comes fragment T^ representing the C-terminal part of the EF-G molecule. The study of the structure of fragments T^-T^ turned out to be simple enough because of their rather low molecular mass. The molecular mass of fragment T g is 6500, that of

- 7500

and that of T,- - 25,000. Using traditional chemical and enzymatic methods for cleavage of the polypeptide chain, we determined completely the primary structure of fragments Tg (4), T^ (6,7) and T,. which all together embrace more than 350 amino acid residues. We have shown earlier (3) that all the fragments of limited EF-G trypsinolysis have arginine as the C-terminal amino acid residue while in the G-factor lysine is the C-terminal amino acid residue. Studying the products of cyanogen bromide cleavage of the whole EF-G molecule we isolated a peptide having

15 the sequence analogous to that of the C-terminal cyanogen bromide peptide of fragment T^ but differing by two amino acid residues at the C-terminus: Gly-Lys. Therefore we concluded that limited trypsinolysis of EF-G resulted in splitting off the C-terminal di-peptide. To obtain overlapping between the fragments of limited trypsinolysis and to elucidate the structure of fragment T^ representing the middle part of the protein polypeptide chain with M r 41,000, we studied the products of cyanogen bromide cleavage of the whole EF-G molecule. As a result, all 24 cyanogen bromide peptides have been isolated, among which 13 peptides constituting the structure of fragment T^. Thus, the study of the structure of fragment T^ amounted to the elucidation of the amino acid sequence of 13 cyanogen bromide peptides and 1. L I M I T E D T R Y P T I C

I T

DIGEST

EF-G

I

T

T

EF-G

FRAGMENTS OF L I M I T E D

A

OF

RG

TRYPSINOLYSIS

RE

RQ

R K

Te

2.

CYANOGEN BROMIDE CLEAVAGE OF E F - G AND FRAGMENTS T ^ AND T5

CYANOGEN BROMIDE

PEPTIDES OVFRLAPPIHG

FRAGMENTS OF L I M I T E D

u 1=1

E

M V

TRYPSINOLYSIS

d

M

M

CYANOGEN BROMIDE P E P T I D E S C O N S T I T U T I N G

THE P O L Y P E P T I D E

E

C H A I N OF FRAGMENTS T(, AND T 5

MG MG \ MEMG M E

diaaauabdddddtiaat

3 MMGG M K M V M E O

SCHEME.

THE PATHWAY OF S T U D Y I N G

MG

ML

THE PRIMARY

STRUCTURE OF

To O B T A I N O V E R L A P P I N G BETWEEN CYANOGEN BROMIDE PRODUCTS OF T H E I R WERE

STUDIED

K

MY

MG

ÜÜ

MKMLMFMER

EF-G.

P E P T I D E S OF FRAGMENTS T^ AND T5

H Y D R O L Y S I S BY T R Y P S I N AND STAPHYI OCOCCUS AUREUS

PROTEASE

16

the search for overlapping between them. To obtain overlapping peptides, we used hydrolysis of fragment T 4 by trypsin and Staphylococcus

aureus protease.

These data pemitted us to represent the partial structure of the elongation factor G. Its polypeptide chain contains over 700 amino acid residues. Several regions of the obtained amino acid sequence have been recently confirmed by the study of the nucleotide sequence of the gene coding for EF-G. Thus, the study of sir operon of E.coli resulted in determination of the nucleotide sequence corresponding to 9 2 amino acid residues from the N-terminus of EF-G (8); elucidation of the nucleotide sequence of the tuf A gene coding for the elongation factor Tu gave the structure of the region of the fus gene coding for the C-terminal part of EF-G

consisting of twenty amino acid residues (9). In both

cases there is a coincidence between the amino acid sequences determined directly and those resulting from studies of the DNA structure. Since EF-G and EF-Tu perform similar functions on the ribosome, i.e. possess ribosome-dependent GTPase activity, it is interesting to compare their amino acid sequences forming the GTPbinding sites. Though the EF-G molecule is almost twice that of EF-Tu, it turned out that in both proteins these regions are located in the N-terminal part of the molecule. The cysteine residue located in close proximity to the GTP-binding site occupies a similar position in both proteins. It is difficult at first glance to find homology between the amino acid sequences of the GTP-binding sites in both proteins. However, if we take into account several insertions or deletions the homology is 30% and if we take into account conservative replacements the homology amounts to 45% (10). These calculations have been made taking into account deletions in 10 amino acid residues in EF-Tu (position 23-32 in the figure). Laursen

17

y

TRYPSIN l(G' (GTP) EF-G

H-N-L

(tRNA)S EF-Tu

1-COOH S(GTP)

VS///A

H-N-l

3 - COOH

TRYPSIN

TRYPSIN B

EF-G EF-Tu

TMD —

W M E Q E Q E R^G I T I

il

il

I II

- TSAATTAFWSGMAKQYE-PHR

II

I I

«

A F D Q I D N P E E K A R G I T I N T S H V 1 1 0 20

m

m

w

I ;

30

v w w w

- I N I

| | ;

I

!

EYDTPTRHYAHV 'tO

GTP-site EF-G

DTDGHVDFTIEVERSMRVLDGAVMV

EF-Tu

D©P

I

III

li

i

¡llliili

60

I t_ a - t uu r n tRNA-site

EF-Tu

VGGVQPQS-ETVWRQA

I

I

II:

I

i:

GHADYVKNMITGAAOMDGAILVVAATDGPMPQTREHLLLGR

Lf—J50

EF-G

y©A

70

VWVWWV/

80

NKYKVPRIAFVNKND

i

QVGVPYI 90

I

lilll

I V F L N Ku|ty»|St Pha Pro Qlu Val Val Gly|Ly»|Thr Val /UpGin •r

r

r

-tr*

z: —

-

r

z

H

Q

Z

H

n

H "

f^-^-

30 40 Arg Glu Tyr Ph« Thr Leu His Tyr Pro Gin Tyr A m Val Tyr Pha Lau Pro Glu Gly -T 4 n.: so •o >0Sar Pro Val Thr Lau Asp Lau|Arg|Tyr AMi|^]val^]val Plia Tyr Aan Pro Gly Thr -T7 -T6 T6T«-T4aCh170 Aan Val Val Aan Hi* Val Pro H ¡a Val Gly T7 __ • -Ch 1-

T1 - T7: tryptic peptides; Ch: chymotryptic peptide; ^ residue identified by automatic Edman degradation; —

residue identified after cleavage with carboxypeptidaseY.

Tryptic cleavage sites are indicated by the boxed amino acids (except T y r ^ ) ; the arrow denotes the chymotrypsin-reactive bond. The chymotryptic peptide Ch1 was obtained by incubation of eglin c with 50 mol % chymotrypsin and fractionation of the incubation mixture by gel chromatography in acidic medium.

Inhibition mechanism In preceding experiments it could be demonstrated that incubation of eglin with varying amounts of chymotrypsin and separation of the mixtures by SDS gel electrophoresis yields a low molecular weight peptide. Figure 2a shows the incubation of eglin c with 5 mol % chymo-

42

Figure 2. Incubation of eglin c with chymotrypsin and separation of the mixture by SDS gel chromatography (in the absence of reducing agents). a) Incubation with 5 mol % chymotrypsin 1

2

3

complex chymotrypsin

- native eglin c ^low molecular peptide

b) Incubation with 50 mol % chymotrypsin

complex chymotrypsin

native eglin c low molecular peptide

x

1 2 3

incubation mixture of eglin c with chymotrypsin native eglin c chymotrypsin

43 trypsin. Three bands appeared: the upper band with a molecular weight of about 30 000 corresponds to the eglin-chymotrypsin complex; the second band with a molecular weight of 8 000 corresponds to native eglin c; and the last band corresponds to the low molecular weight peptide. The same band pattern is obtained from the incubation mixture of eglin c with 50 mol % enzyme (Figure 2b). The only difference is that the intensity of the eglin band is decreased whereas the intensity of the complex and the low molecular weight peptide band is increased. From sequence data we could identify the low molecular weight peptide as the C-terminal fragment of eglin c comprising positions 46 - 70 (Ch1). From these results it seems likely that during complex formation of eglin c with chymotrypsin limited proteolysis occurs resulting in the release of a C-terminal peptide. The N-terminal part of the inhibitor molecule remains firmly bound to the enzyme in the complex. This complex might be covalently linked as it does not dissociate in SDS gel electrophoresis or during gel chromatography in acidic medium. Generally, proteinase inhibitor proteins do not form covalent complexes with proteinases. They react according to the following mechanism: E + I

kU

—1

L

k_ —2

k-3 C r ^ A -3

k. -4

L* ,

kr 3

» E + I*

-5

E - enzyme; I - native inhibitor; I* - modified inhibitor (reactive site peptide bond hydrolysed); L and L* loose complexes consisting of enzyme and native inhibitor or modified inhibitor, respectively; C - stable enzyme-inhibitor complex; A - acyl-enzyme-complex. Inhibitor and enzyme form via a loose intermediate (L) the noncovalent, catalytically inactive complex C (2). This complex is very stable as expressed by a low dissociation constant. Limited proteolysis, i.e. hydrolysis of the reactive

44 site peptide bond of the inhibitor may occur resulting in the formation of the modified inhibitor. The modified inhibitor is also inhibitory active, although the velocity of complex formation is normally reduced. It may be transformed again to native inhibitor by thermodynamically controlled resynthesis of the cleaved peptide bond. All inhibitors shown so far to obey the above given standard ii

mechanism are characterized by a reactive peptide bond which is encompassed in at least one disulfide loop. This disulfide loop ensures that during conversion of virgin to modified inhibitor the two peptide chains cannot dissociate" (3). However, the eglins do not contain a disulfide bridge, i.e. there are characteristic differences between the eglins and the "standard" proteinase inhibitor proteins: (i) The enzyme-inhibitor complex formed by the eglins seems to be a covalent complex, the one formed by the standard inhibitors is a noncovalent complex. (ii) Complex formation is reversible for standard inhibitors but obviously not for the eglins. (iii) The degree of limited proteolysis of the eglin reactive site peptide bond correlates with the applied enzyme concentration but depends on a true thermodynamic equilibrium between native and modified inhibitor for the standard inhibitors. Thus, the inhibition mechanism of the eglins resemble that of plasma inhibitor proteins, e.g. °Gal 3 ^0^

3Gal

3. 6Man

|3 4GlcN

6| 4GlcN

ASN

J Fuc

2Man ^

Structure of glycopeptides of the complex type NA

3 G a l — 4GlcN

NA

3Gal — 4 G l c N —

3. 6Man — 4GlcN — 4GlcN — ASN 2Man^

virus. Paramyxoviruses are efficiently propagated only in certain epidermal cells, but not in fibroblast

cells such as BHK-21

which apparently are unable to terminate the carbohydrate structure by acetalization. This terminal portion seems to be necessary for maturation of those viruses which contain a neuraminidase activity. The viral neuraminidase has been shown to function intracellularly (9) and may interfere with the release of the virus from those host cells which terminate the carbohydrate structure only by neuraminic acid and not by acetaldehyde. A scheme of paramyxovirus maturation is shown in Fig. 7. The viral polypeptides are synthesized in the endoplasmatic reticulum, glycosylated in the Golgi and terminated by acetaldehyde on their way to the cell membrane. The viruses are liberated by budding from the plasma membrane • The infectious cycle is again initiated by adsorption through the hemagglutinating activity of the HN protein to uninfected host cells presenting neuraminic acid residues on their surface.

59 viral neuraminidase

ù

ò

.

Golgi HN transferase

F

celi membrane

QCetQldehyde

Fig.7. Maturation of Paramyxovirus SV5.

References 1.

Choppin, P.W., Compans, R.W.: Comprehensive Virology (Fraenkel-Conrat,K., Wagner, R.R., eds.) Vol 4, 95-178 (1975).

2. 3.

Scheid,A. , Choppin, P . V I . : Virology 62., 125-133 (1974). Fukami, Y. Hosaka, Y. Yamamoto, K.: Febs. Lett. 114, 342-346 (1980). Prehm, P., Scheid, A., Choppin, P.W.: J. Biol. Chem.254, 9669-9677 (1979).

4. 5.

Laver, W.G.: Virology 45_, 275-288 (1971).

6. 7. 8. 9.

Klingenberg, M.: Nature 290, 449-454 (1981). Prehm, P. Scheid,A.: J. Chromatogr. 166, 461-457 (1978). Prehm, P.: Carbohydr. Res. 78, 372-374 (1980). Merz, D.C., Prehm, P., Scheid, A., Choppin, P.T'7.: Virology 11 2, 296-305 (1981).

HAEMOGLOBIN Primary

POLYMORPHISM

IN C H I R O N O M U S

Structure, Allergenic

(DIPTERA)

Determinants

and

Monomer-Dimer

Equilibrium

Heinz Aschauer, Traute Kleinschmidt, Wolfgang Steer, Braunitzer Max-Planck-Institut

Gerhard

für Biochemie, D-8033 Martinsried

b.München

Introduction

The

family

There

Chironomidae

are s o m e

in E u r o p e

75 000

alone

over

(midges)

species 1 500

stage. The These most

haemoglobin

upon

haemoglobins

approximately

electrophoresis

been c h a r a c t e r i z e d

species and

depending

population

interesting tionary

of one thummi

(4). This

a structural,

2-6 % d e -

high

factors.

in c o n t r a s t

species

12 d i f f e r e n t stage

form

can be

components

and

000

separated

(Fig.

1).

In

haemoglobins

is f o u n d

in all

environmental

fuctional, genetic

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter & Co., Berlin • N e w York

of

mono-

d e g r e e of p o l y m o r p h i s m

standpoint.

to

weights

16 000 a n d 32

polymorphism

upon developmental This

of

and

(1).

larval

between

haemoglobins

weights

into m a n y

thummi

conditions.

from

in the

have m o l e c u l a r

Chironomus

with molecular

the c a s e of C h i r o n o m u s

varies

described

and environmental

which

Diptera.

the w h o l e w o r l d been

low m o l e c u l a r w e i g h t s ,

haemoglobins

( 2 , 3 ) . The h a e m o g l o b i n

in d i s c gel have

concentration

10® d a l t o n s .

mers and dimers daltons

have

have

haemoglobin

s t a g e of d e v e l o p m e n t

invertebrate

to the o r d e r

spread over

species

M o s t of t h e m h a v e e x t r a c e l l u l a r pending

belongs

and

evolu-

is

62

IA Fig. 1 up HI.mo 7IIA IT YI "STUB YIII IX X

1 The

presence

of

giant

Chironomus

enabled,

structural

gene

sis

pattern

parent gene

localized

lized

to p a r t i c u l a r

existance

indicates through

of

that

the

gene

Chironomus

The

12

haemoglobins

in the

first

time,

Comparison with

of

polyare

haemoglobin

polymorphism

gel

chromosome that

structure III)

were

on o n e

o u t of a

even

loca-

chromosome.

single

of h a e m o g l o b i n

of

haemoglobin

of b a n d s o n t h a t

locus

of of a

electrophore-

the

(chromosome

components

or g r o u p s

glands

localization

disc

showed

chromosome

haemoglobin

salivary the

the

the

generations,

bands

the

the

of

thummi.

of

i ndi c a t e d .

on o n e

Single

electrophoresis

haemolymph

morphic

of h a e m o g l o b i n s

of f o u r .

gel

thummi

chromosomes

(5,6).

total The

the

for

and hybridized

was

Disc

chromosome

probalby

evolved

duplication.

Results The

sequences

thummi and

have

CTT

of all

been

IA a r e All

globins

represent

2,3

acids.

and

4.

determined.

identical

mersim. amino

12 h a e m o g l o b i n s

other

11 the

The

primary

and here we

haemoglobins partial

of

thummi

structures

suspect

are

determination

Significant

of C h i r o n o m u s

structural

different. the

sequences

of

The

sequence are

shown

12 of in

CTT I isohaemo1738 figures

64

65

Uí C •H m £ o C 3 t/1 eu c S- S-C 0) 1— 00 =3 0) ai JZ —i Q_ IO 3 i— ai _J c

>i CD c 5 ai

VI

>>

3 3 3 CD CD CD Í. i. i. aj ai a j oo 00 00 3 3 a> ai aj CL _j ai ai ai ai ai JZ JZ . c ^ CL es- O- CL o . 1- >> >1 s- tai aj a j CD oo 00 00 CD rl. ,-iIO IO IO IO IO > > > > >

(/I

3

>>

—I S- 1ai aj oo 00 a> ai jz .c a. a. 1 i/i >1 « ï CD "C eu CD ,-iIO aj i— « •— IO IO > i—i > > < rl à) ai —i i—i 1—1 > > 1 t/1 en t/1 OÌ t/1 >) >1 Î. >1 < —I > C c t/l 00 >> —I cd < 1 IO IO 10 io cd c «C c < m V) c oo co r e CD 31 n : >i to Q. s - ' a i IO 00 i— 1— 5 > (U 00 CD oo ,1 ai ai 0) ai io . c . e -C jz > a . CL O. Q. a. 3 o CL c r— t/1 r— s- t/1 CD a . < CD c IO o 10 18 10 l« t a .

3

CD CS L ai ai oo 00 3 IO ai >

aj *—i en en SC < >i s. •C 1— IO IO ai i—i

c oo m s. 1— 10 r— =c ai JC Cl. C CD

i—

ai

>. >.

CD 1

CD 1

> > t— 1— 1— 1— t— 1— 1— 1— c j (_> CJ CJ

ca. i—i »—« »—i > hhCJ

ai

Ol O) Oi s_ t- sc c < s_ > > i1— .c l'- 1— CD io 10 ia) «t < « t oo i/i t/1 00 •r— in • I n : a : Si. t. .c IO l'- 1— 1— > io 10 IO IO 1— i— r— 1— c 1 CD _i a) E •H Q

aj i—i

3

3

CD CD Sai co CD a> a; i— sz CL

>,

ai sz Cl >>

ai sz CL

>,

CD CD rl. ai IO i— >

1

ai

O) i. < >1 iai oo IO IO D1




hIO r— C ai QIO r—

JZ CL a. 00

>>

IO


1-1

rW ai

.c -p

o c ai 3 CT

•H fa

66 A comparison that

the

varies

of

sions

of

(Fig.

136

up to

insertions

20

positions

components.

There

structures

relatively residues.

acids are

and

also

and deletions in w h i c h

If any

idenitiy. CD

are

- 151

6 amino

2).

as

in t h e

primary

differences

between

acids

50%

the

Despite

this

segment, which

large. There

high

chain

of

is the

up to

2

same

exten-

amino as

There

well

are

in all

there

variability

is p r a t i c a l l y

length

extensions

compared

shows

N-terminal

the c h a i n s .

acid

are

globins

The

are

deletions

within

chains

the

C-terminal

the amino

two

of

only

12

is a b o u t

there

is a

a

region,

constant:

CD

D

-Phe-Pro-Glu-Phe-Ala-Gly-Lys-Asp-LeuThe of

reason

for

this

the m o n o m e r

known

The

haem the

in the

show

has

tidine

normal

E7 on

the

globins,

does

the

in the

iron

pionic

acid

histidine

The

Chironomus

Allergic insect or

larvae,

(RAST)

Chironomus

180°

o u t of

the

around

The

histidine

up

present a salt distal

its

bulky

by

detect

thummi

How-

haemoof

the

is b o u n d

to

distal

in all

CTT

haemo-

position

side side

with is

hisof

a

pro-

residue

chain,

pushes

pocket.

is a p o t e n t

in p e o p l e the

IV

10%

bridge

the

very

known

axis.

F 8 . The

On

haem

to

haem

is

myoglobins.

and CTT

of

the

and

coordination

forms

is

t o the well

haem.

laboratories.

we c o u l d

(8,9).

sixth

but

III that

forms,

significant

the a,y meso

CTT

structure

structure are

haemoglobins

because

occur

either

compared

X-ray

liganded

there

indicates

the

The

tertiary

hand, although

haemoglobin

thummi

as

proximal

form

which,

reactions

in r e s e a r c h

Test

deoxy of

The

However,

orientation the

clear.

in v a r i o u s

on m o n o m e r i c

other

group

the

(7).

which

not occupy

isoleucine

III,

complex

is t u r n e d

through

not

vertebrate

a signal

its

protein

E11,

haem

of t h e

group

is

of m y o g l o b i n .

NMR m e a s u r e m e n t s

globins

CTT

A resoltuion

complexes haem

ever

o

to t h a t

differences haem

component

at a 1,4

similar

constance

antigen

who

have

preparation Using

a

specific

haemoglobins

in m a n

(10).

had contact

and

use

of

fish

with food

Radio-A11ergo-SorbentIg

E antibodies

(CTT

IA w a s

not

against investi-

67 gated). active

It w a s

also

shown

that

when

a larvae

extract

The

allergic

action

graphy. nents

was

also

dimer

CTT

VI, a region was

antigenic dues

63

vity. be

determinant

- 98

terminal

confirmed

(E11

One

in t h e

could

is

(F2)

separated

of the

test.

expect

of t h e

cleavage

of

F1

90

the

this

haemoglobins

dimers.

The

of

dimers

haemoglobin

Chironomus can

thummi

dissociate,

concentration

and

least

contains

region

loss and

chromato-

at t h e

the

form

monomers

upon

the

(Fig.

5)

(11,12).

• CO

o2 x Met "

Q. o 2,

u

Fig.

5-

Monomer-dimer component

X of

its d e p e n d e n c e

7.0

equilibrium Chironomus upon

pH and

S.O

of

to

.

thummi

ligand

6.0

acti-

depending

o.

5.0

C-

sequence:

• CAT

e.,0

resi-

determinant

have

the

one

of a n t i g e n i c

3.

1,

compo-

component, at

antigenic

point

Leu-Ala-Lys-Glu-Leu-AlaThe

In o n e

region

complete

gel

were

haemoglobin

in w h i c h

This

Cleavage

caused

fractions

using

separated

identified

present.

therefore

region

was

haemoglobin

by a s k i n

- FG2).

of Lys 89

the

the

thummi

pH

haemoglobin thummi

ligand.

and

pH

and

value,

68 Concerning different The

CTT

until 7.0. an

for

pH-dependence each

pH 8 - 9 , w h i l e The

further

the

all

other

in t h i s

a monomer

after

bridge

t h a t .forms a s a l t

IIB

they

are

should

Monod

(16).

as m o n o m e r s . all

of

lysine

and

glutamic This

from

acid

by

thereby

has an

This would

G7

suspicion

is the

identical, the

isologous

is s t r e n g t h e n e d

the monomer

components

no g l u t a m i c

acid

at

also

by

have

maleic

the

have as

anhy-

effect stability

t h e d i m e r s of C T T

we

in the

hae-

subunits,

as d e s c r i b e d believe

salt

the o b s e r v a t i o n histidine

po-

residue

numbered

structure,

partner

dimer

In

important

association

primary

a

dimers

is a

explain

even

of

11B e x i s t e d

with

(His, Lys)

isologous

comparing

other CTT

residues

G19

sites. role.

diethylas

present

position

be f o r m e d By

with

whereas

only

pH

gives

modified.

were

reaction.

formed

chains

remained

of the CTT II/3 d i m e r a t h i g h pH v a l u e s . A s moglobins

side

(15) w e r e

a lysine

bridge

dimerisation

up to

important

residues

CTT

form

the binding an

the

position.Conversely, that

cyanomet

equilibrium

of

play

of h i s t i d i n e

reaction

We believe

the

salt

that only

11B h a s

histidine

on

to t h e n a t u r e

compoenents

G19, CTT

dride.

found

is

is a d i m e r

(14) and a r g i n i n e

reaction we

in the

X component

interactions

the

it

component.

as a d i m e r

the m o n o m e r - d i m e r

as

ionic

investigate

selective

sition

CTT of

(13), lysine

pyrocarbonate, while

the

indication

that

histidine After

exists

pH d e p e n d e n c e

important

of t h e e q u i l i b r i u m ,

haemoglobin

11B c o m p o n e n t

We a s s u m e To

the

by that

bridge.

that

some

in p o s i t i o n

G19

of but

G7.

Discussion In o u r m o d e l , larities but

not

to

in the

ly, through units salt

one

the

in the bridges

inclination

can

see,

binding

tetramer in the the

regions.

formation,

direction of

in t h e q u a r t e r n a r y

6 - B or a - a s u b u n i t s

CTT

of

the

subunits

However in t h e

G helix

dimers and

structure,

of m a m m a l i a n

in t h i s

they

differ

inclination (17,18). region

therefore

simi-

haemoglobins, considerabof

the

Assuming are

tetramer

sub-

that

formed,

an

formation

69 is

impossible

Goodman, the They

Moore

heterotetramer further

ß chains

evolved

thought

that

of m a m m a l i a n

a chains can homodimers the

arid M a t s u d a

form

deduced via

a preceding homotetramer

homotetramers.

homotetramers

and

phylogenetic

the

haemoglobin

of C h i r o n o m u s

from

a still

homotetramer

as o n l y Have we

haemoglobins, earlier

data

was

(19).

similar

ß chains

to

and

discovered,

a further

that the

not

in

the

ancestor

developmental

form

of of

haemoglobi n ? The

phylogenetic

ration must

of

have

the

insect

taken

place

million 400 ship

million

dimers cestors

the

and ago.

sites.

thummi

of the m a m m a l i a n

binding

the

dimer

sites

binding

therefore

have sites

not able

others.

sepa-

s e p a r a t ion

years

of m y o g l o b i n s into

we

ago,

thummi

f i n d no

we

different into

that

500

ß chains

assume

haemoglobins

no p h y l o g e n e t i c to e v o l v e

about

a and

could

Therefore

heterotetramer have

early

This

650 m i l l i o n

separation

In o u r w o r k

binding

a very

f o r m a t i o n of h e m a t e t r a m e r s

separation the

shows

from

approximately

the

of C h i r o n o m u s

the were

ago

years

between

haemoglobins

the accepted

between

years

of h a e m o g l o b i n

place

t h a t is, b e f o r e took

tree

are

relationthat not

haemoglobins relationship,

the an-

and

that

Further

characteristics tetrameric

about

and

haemoglobins.

References 1.

T h i e n e m a n n , A . , in: Die m u s , S c h w e i z e r b a r t 1 sehe 1954

Binnengewässer, Vol. Verlagsbuchhandlung,

2.

Svedberg, T., 1700 ( 1 9 3 4 ) .

3.

Braunitzer, G., Braun, 3 4 0 , 80 ( 1 9 6 5 ) .

4.

Braun, V., Chrichton, R.R., Braunitzer, Z. P h y s i o l . C h e m . 3 4 9 , 197 ( 1 9 6 8 ) .

5.

Tichy,

H.:

Chromosoma

6.

Tichy,

H.:

J. M o l .

7.

Steigemann,

E r i k s o n - Q u e n s e l , I.B; J . A m . V.:

Hoppe-Sey1 er's

(Berlin)

Evol . 6 , 39

W., Weber,

29,

131

XX: C h i r o n o Stuttgart,

Chem. Z.

G.:

S o c , 5_6

Physiol.

Chem,

Hoppe-Sey1 er's

(1970).

( 1 975).

E.: J. M o l .

B i o l . J_27, 309

(1979).

70 8.

R i b b i n g , W. , K r ü m p e l m a n n , 9 2 , 105 (1978).

D.,

Rüterjans,

H.:

FEBS

9.

La M a r , G . N . , S m i t h , K . M . , G e r s o n d e , K . , S i c k , k a m p , M . : J. B i o l . C h e m . 2J55, 66 ( 1 9 7 9 ) .

Letters

H.,

Over-

10. B a u r , X . , Z i e g l e r , D . , R e i c h e n b a c h - K l i n k e , H . H . , H., B r a u n i t z e r , G.: Naturwiss. 365 (1980).

Aschauer,

11. B e h l k e ,

J., Scheler,

(1967).

12. B e h l k e , Müller, 34, 365

J., Pfeil, W., Blanck, J., Ristan, 0., Rein, H., K . , M ö h r , P . , S c h e l e r , W . : A c t a B i o l . M e d . Ger(1975).

W.:

Fahrney,

J. B i o c h e m .

3,

152

13. M e l c h i o r ,

W.B.,

D.:

Biochemistry

9 , 251

14.

D., Laskowski , M., Jr.:

Biochemistry

1 1 , 3451

Kowalski, ( 1 972 ) .

Jr.,

Eur.

15. D i e t l , T . , T s c h e s c h e , 357 , 6 5 7 ( 1 976 ). 16. M o n o d , J . , W y m a n , 88 (1965). 17. P e r u t z , 18.

M.F.:

H.:

H o p p e - S e y l e r ' s Z. P h y s i o l .

J., Changeux,

Nature

228,

726

J.P.:

J. M o l .

Biol.

Chem. 12, -'

(1970).

Perutz, M.F., Rossmann, M.G., Gullis, A.F., Muirhead, W i l l , G . , W o r t h , A . C . T . : N a t u r e J_85, 416 ( 1 960 ).

19. G o o d m a n , M . , M o o r e , (1975).

( 1 970).

G.W. Matsuda,

G.:

Nature

253,

603

H.,

HIGH

ALTITUDE

RESPIRATION

INDICUS)

AND

THE

IN A V I A N

HEMOGLOBINS

Investigations anser)

DIFFERENT

OF THE

on h e m o g l o b i n s

(1), Bar-headed

Goose

and

BAR-HEADED

EVOLUTION

of t h e (Anser

(Branta

canadensis)

Ostrich

Gerhard

Braunitzer

and

Walter

Max-Planck-Insitut D-8033 Martinsried

für bei

Biochemie München

GOOSE

OF THE a -

Grey-Lag indicus),

(Struthio

(ANSER

AND

g-CHAINS

Goose

(Anser

Canada

Goose

camel us)

Oberthür

Introduction

We

have

investigated

headed Goose

(Anser

structure

of

the

Lag

(Anser

Goose

Canada To and

Goose

determine g-chains

complete

were

amino

high

indicus)

altitude and

ma in c o m p o n e n t s anser),

(Branta the

the

fragmented

acid

sequences

and of

of

the

(Anser

Ostrich the

of t h e

the

hemoglobin

Goose

and

structure

described

the

Bar-headed

canadensis)

primary

have of

respiration

primary of t h e

products

Grey-

indicus),

(Struthio

hemoglobins,

the a - and

Bar-

isolated.

6-chains

camelus). the

a-

The

were

de-

A b b r e v i ati o n s : Q u a d r o l = N ,N ,N ' ,N ' - T e t r a k i s ( 2 - h y d r o x y p r o p y l ) e t h y l e n d i a m i n e , Reagent I = sodium 1 -(isothiocyanato)benzenesulphonate, R e a g e n t IV = t r i s o d i u m 7 - ( i s o t h i o c y a n a t o ) n a p h t h a i e n e - 1 ,3 ,5tri s u l p h o n a t e , IPP = m y o i n o s i t o i - 1 , 3 , 4 , 5 , 6 - p e n t a p h o s p h a t e , DPG = 2 , 3 - d i p h o s p h o g l y c e r a t e .

C h e m i s t r y of P e p t i d e s a n d P r o t e i n s , V o l . 1 © 1982 by W a l t e r d e G r u y t e r &. Co., B e r l i n • N e w Y o r k

72 termined

by a u t o m a t i c

sequencing

of the intact chains and

lated f r a g m e n t s . The d i f f e r e n t e v o l u t i o n and the problem of high a l t i t u d e Goose are d i s c u s s e d

of the a -

breathing

of the

and

iso-

B-chains

Bar-headed

(2,3).

Methods Crude h e m o g l o b i n moving

was p r e p a r e d by lysing e r y t h r o c y t e s

cell m e m b r a n e s

g r o u p , the a -

ion e x c h a n g e

CM-52

(2)).

using

chromatography

tic d i g e s t i o n

m e t h o d s as d e s c r i b e d e a r l i e r

maleic anhydride

the e - a m i n o - g r o u p s

(5); acid h y d r o l y s i s

d i g e s t i o n with S t a p h y l o c o c c u s Amino acid a n a l y s i s :

isolated

isolated (2,3):

cya-

(4); tryptic d i g e s t i o n and l i m i t e d

after blocking

aureus

of lysine

of A s p - P r o - b o n d s protease

V8

(6);

(Palo A l t o , C a l i f . ,

a Beckman USA).

Amino acid s e q u e n c e a n a l y s i s : amino acid s e q u e n c e s were mined automatically

in a Beckman

S e q u e n a t o r , Model

Lysin peptides

(0,5 pmol) were c o u p l e d with 8 pnol

(9) or r e a g e n t

IV

Arginine

minutes

using

3 M trifluoracetic

ti on HPLC

programme. Sequenator

acid at 80° C for

e i t h e r thin layer c h r o m a t o g r a p h y

(13).

of r e a g e n t I

from the

and the resulting p h e n y l t h i o h y d a n t o i n

tified using

(8).

a 1 M diethyl a m i n o p r o -

(11,12). Amino acid d e r i v a t i v e s

were c o n v e r t e d

deter-

890 C

(10) and s e q u e n c e d using a Quadrol

peptides were s e q u e n c e d using

pine buffer

trypwith

(7).

a n a l y s e s were c a r r i e d out using

121 C amino acid a n a l y s e r

heme-

CM-cellulose((Servacel

for s e q u e n c e a n a l y s i s were p r e p a r e d and

the following

nogen bromide c l e a v a g e

Model

on

re-

off the

and B - c h a i n s of the main c o m p o n e n t were

using

Fragments

and n u c l e i . After cleaving

and

amino acids

14 iden-

or g r a d i e n t el u -

73 Results

and

Discussion

Biology

of the G e e s e

The Grey-Lag

Goose

ser

belong

indicus)

da G o o s e the

family

portant geese The

place

of

Goose

northern

areas

breeds

Goose

African

ber

of t h e

on the

in

the

im-

(14)

of t h e

existing

areas

of the

Boreal

area

of the

Grey-

from

Iceland

In E u r o p e

they

breed mostly

In c o n t r a s t ,

in c e n t r a l

goose

Cana-

geese

stretching

cost.

They

(An-

period.

to w i n t e r

common

(see

to J o h n s o n

evolution

Goose

g e e s e ) , the

Branta

the breeding

lakes

Himalaya

(real

in the m i l d e r

Today zone

subspecies.

in

India

in A m e r i c a

inhabit,

the

Asia

in

Bar-headed

(4000-6000

(15-17).

and gives

like

through

The

Canada

rise

the G r e y - L a g

m) to

Goose,

Ostrich

(Struthio

camelus)

suborder

Ostriches

( S t r u t h i o n e s , f a m i 1y Struthionidae)

since

but

According

to S a l a c h i n . and

relatives

Africa

Anser

Bar-headed

the g e n u s

in the

lives

the

areas.

The

whose

(1)

a wide

is the m o s t

low-lying

steps

on h i g h l a n d the

of t h e

bird

Goose

covers

and crosses most

(ducks).

to

at the T e r t i a r y

Eurasia

low-lying Goose

and

the the genus

Palaearctic.

the

Ostrich

anser)

canadensis)

Anatidae

took

of the

(Anser

phylogenetic

Grey-Lag

Zone Lag

(Branta

and

have

the

its

inhabited

Eocene

position

period.

in the

large

areas

Today

family

is the o n l y of A s i a ,

it is the

tree

living Europe

largest

of b i r d s

is

memand

living

still

di s p u t e d .

Physiology In all

of

birds

hemoglobins

the A v i a n (18,19)

(ca.

IPP

5 pmol)

DPG a n d o t h e r o r g a n i c ty of o x y g e n Goose only

small

ween

the

is the m a j o r together

phosphates.

for h e m o g l o b i n s

and Canada

differences

Hemoglobins.

Goose

differences became

PcnO?

for

with

allosteric low

Investigations

in the G r e y - L a g

showed,

b u t on the

addition

significant

Canada

and Bar-headed

of A T P , on

Goose,

in t h e a b s e n c e

highly

effector

levels

of

of

ADP,

affini-

Bar-headed

phosphate,

phosphate

(20). The Goose

the

of

the

difference hemoglobins,

bet-

74 without phate

p h o s p h a t e , was

12 t o r r . T h e

a highland gen

bird,

affinity.

Grey-Lag

ca.

P5Q02

is 2 9 . 7

Geese

have

results

correlate

and C a m e l ,

Primary

with foetal

v a 1 u e s

lower

of

presence

of the

indicates

the b l o o d of the 39

oxygen

and adult

-

5

acid

of h e m o g l o b i n s

sequence

of the a - a n d

and Struthio

Comparing

the h o m o l o g y

including

chicken

a very

lowland

high

oxyand

torr

affinity

Goose,

Canada

(20).

respecThese

on h e m o g l o b i n s

from

the

(21).

from Anser camelus

g-chains

are g i v e n

of the h e m o g l o b i n

(22,23)

of the

anser, Anser

the f o l l o w i n g

in f i g u r e s chains

major

i n d i c u s , Branta 1 to

4.

of v a r i o u s birds,

differences

can

be

seen:

1: A

B

Amino acids

Nucleotides

a

ß

tx/0

a

ß

a/ß

GG/BG

3

1

3

3

1

3

CG/BG

5

3

1 .67

6

3

2

CG/GG

2

3

0 .67

3

3

1

OS/GG

15

4

3 .75

16

4

4

OS/BG

16

5

3 .2

1 7

5

3,.4

OS/CG

16

6

2 .67

1 7

6

2 .84 ,

CH/GG

30

6

5

39

6

6,.5

CH/BG

32

7

4 .71

41

7

5,.86

CH/CG

32

5

6 .4

41

5

8,.2

CH/OS

29

7

4 .15

39

7

5,.57

CH = CG =

phos-

Bar-headed

a n d

humans

of

Structures

component

Table

in the

blood

investigations

The amino canadensis

but

torr which

P5o°2

indicating a m u c h

The

for the

In c o n t r a s t ,

tively, Llama

2 torr

C h i c k e n , GG = G r e y - L a g G o o s e , C a n a d a G o o s e , OS = O s t r i c h

In A the n u m b e r corresponding

of a m i n o

number

BG

;

= Bar - h e a d e d G o o s e ,

acid exchanges

of n u c l e o t i d e

are

shown^.

exchanges,

in B the

assuming

a

mini-

75 mum

of b a s e

changes, and

exchanges.

the

For

relationship

3-chains

both

between

is c a l c u l a t e d

terminal

region,

In t h e

half

constant.

helices.

region.

Three

gions

and

total

mutations

the

Phosphate

the

and

of the m u t a t i o n s the

of the m u t a t i o n s

There

are

no s u b s t i t u t i o n s

are

present

Binding

found

Sites

in t h e

H helix.

in the

and

are

a-

in t h e

N-

C-terminal

is

contain

mutations are

in the

ex-

a/6.

of the a - c h a i n s

beginning of the

nucleotide

differences

of t h e c h a i n w h e r e a s None

IJ-chains, t h e m a j o r i t y

N-terminal

acid

as a f a c t o r

In the a - c h a i n s , the m a j o r i t y relatively

amino

tryptophan.

also

lie

in the

in

D to G

interhelical

Only

very

the

few

re-

of

the

the a -

and

G-chains.

Unequal

Evolution

of

S-Chains The

oxygen

(18),

in- b i r d s

paring amino all

now

of the

have

been

sites

human

exchange

was

was

somewhat

by c o m p a r i n g

of

rate

five

b i r d s , we

using

availability

this

the m u t a t i o n

higher

than

primary have

in t h e 6 - c h a i n s

one e x p l a i n

constructed

the

pal a e o n t o l o g i c a l

to be m a d e .

data

of

result? and

primary

enables

hemoglobin, been

finds

of b i r d s

very

rare,

paleontologists

family

tree.

The

example, million

are

the c h i c k e n years

old.

and

the

lower

common

muare

structural

data.

compa-

rate

( 2 5 ) . As

of

can

say

very

of,

for

to be

of 6 m i l l i o n

one

fossil

ancestor

is t h o u g h t

rate

trees

a mutation

calculated

goose,

By a m u t a t i o n

a-chain.

quantitative

T h u s , for

the

8-chain

the a - a n d 13-

Family

years

about

in the

a significantly

6-10 million

little

In

case

subtstitutions

of t h e

every

has

comthe

table).

surprising

of s e q u e n c e

in one

rate

that

by

hemoglobin.

no

structure

found

(see

phosphate

(24) w i t h

(except

reported)

that

by

determined

3 - c h a i n of c h i c k e n

investigated

thought

were

hemoglobin

it w a s

tation

risons

of

positions.

chains can

binding

is c o n t r o l l e d

fourid in t h e s e

hemoglobin

However,

The

The

sequence that

hemoglobins

analysis

a conservative been

Until

How

of

IPP.

X-ray

acid

where

of

the

birds

have

affinity

80-120

years,

this

76 would mean changes

a normal

a significantly rate.

site

stead

of 6 as

rates

of e v o l u t i o n

fixes

the

structure

(23).

The

rate

therefore

The can

normally

oxygen

compared

the m o s t This

surprising

with

M.

structure

there

these its

Bar-headed

different for

IPP

which

12 b i n d i n g

sites

be r e d u c e d sites

and

in

will

alteration

birds. is no

exchange

The

the

room

of the to the

E12

Val

hemo-

side

found)

alanine

in t h e

chain

therefore

dis-

occupied

Ser w a s

of t h i s

as

Goose

of

is

it

ex-

of a w r i t t e n

a63

B2 so t h a t

a longer

for

Goose as

the

Bar-headed structure

group

a21

Canada sites,

and H b A / H b F ,

rabbit where

B-methyl for

and

binding

Bar-headed

the m u t a t i o n s

position

of a l a n i n e

of A l a

Goose

is a r e s u l t

"The

of t h e

Llama/Camel Of all

finding

Goose

hemoglobin

phosphate

in t h e

(except

C=0 c a r b o n

The

increased

(12 m o l e c u l e s i n -

requirement

Grey-Lag

found.

F. P e r u t z .

mammals

other

the

that

ex-

mutation

be t h e

no b i n d i n g

in t h e a - c h a i n

drastic

cussion

sition.

of the

were

globin.

in all

could

B - c h a i n will

has

of t h e

by d i f f e r e n t

(E1 2 ) Val

in all

an a b n o r m a l

through

of the

of t h e

case, for e x a m p l e , a63

the

B-chain

affinity

no d i f f e r e n c e s

touches

by

(2 x 15 however,

evolve.

to t h o s e

causes

this

B-chain,

hemoglobins

We b e l i e v e

of t h e

Respiration

change

and

for

caused

of e v o l u t i o n

n o t be c a u s e d

by A l a

therefore

in a v i a n

are

for the a - c h a i n

for the

to t h e a - c h a i n w h i c h

higher

is the here

and

in m a m m a l s ) .

Altitude

Goose

rate but

explanation

binding

comparison

years)

reduced,

A possible

phosphate

High

mutation

= 90 m i l l i o n

normal

in t h i s

causes

the

po-

heli-

o

ces a

E andB

large

to

be

pushed

alteration

at

in t h e

be n o t e d

histidine

of t h e heme c o m p l e x .

B binding

above,

probably

hemoglobin pretation Goose.

sites,

when of

the

IPP

the

E-helix

a different

is b o u n d

high

with and

altitude

represents

structure." in E7 c a r r i e s

Additional

together

cause

1.5 A a p a r t w h i c h

tertiary

It s h o u l d and

that

least

the

mutations

allows

respiration

distal

at the

substitution

conformational

thus

the

change

a molecular

of t h e

a

described in inter-

Bar-headed

77

i/t

r—

0>

«— r— ^

_Q O CD O

— < < o

< Ü3 U
>

ai t. CD "O e C - Q

+

80 z

377

—C=0I m/e 346

6

Ph-C-Ph Ph m/e 243

(- 60

^ ^

378

346

& 40

243 244

20

200

250

400 m/e

350

300

Figure 2. F.d. mass spectrum of H-Cys(Trt)-OCH^ and interpreted fragmentation at 6 mA e.h.c.; solvent: DMSO.

CH3(

m/e 309 ( M H * = 310)

NH-CH-C0(0H

m/e 210 +

nhj°\J

nu OH

nu OH II

Fig. 5.

0

HOOC-CH2-CHJ-CH-COOH NH

J

I H2N4H

nu OH

.0 T

h

° ! OH

' OH

III

I: nucleoside of nikkomycin X; II: peptidenucleoside (nikkomycin N) of n i k k o m y c i n J; III: peptide-nucleoside (nikkomycin M) of n i k k o m y c i n I.

F i n a l l y nikkomycin B was isolated (5) in w h i c h the pyridine ring of nikkomycin X is replaced lay a phenyl ring. The fungicide activity of this compound is rather low compared to n i k k o m y c i n X. Since nikkomycin X and Z are p r o d u c e d in amounts up to culture filtrate

kg/l

and due to their h i g h fungicide and insec-

ticide activity they are promising compounds for practical application in pest control.

References 1.

Dahn, U., Hagenmaier, H. , Höhne, H., König, W . A . , W o l f , G . , Zähner,H.: Arch. Microhiol. 107, 1^3 (1976).

2.

Ishikawa, K., Achiwa, K., Yamada, S.I.: Chem. Pharm. Bull. 19, 926 (1971).

3.

König, V.A., Pfaff, K.P., Bartsch, H . H . , Schmalle, H., Hagenmaier, H.: Liehigs Ann. Chem. 1980, 1728.

k.

Hagenmaier, H., Keckeisen, A . , Dehler, W . , Fiedler, H.P., Zähner, H., König, W.A.: Liehigs Ann. Chem. 1981, 1018.

5.

König, W . A . , Hass, W . , Dehler, W . , Fiedler, H.P., Zähner, H.: Liehigs Ann. Chem. 1980, 6 2 2 .

Peptide Syntheses. Biological Activity A n a l y t i c a l P r o b l e m s of S y n t h e t i c

and

Peptides

ELECTROCHEMICAL INTRODUCTION AND SELECTIVE REMOVAL OF A NEW TYPE OF AMINO- AND CARBOXY-PROTECTING GROUP

FOR PEPTIDE SYN-

THESIS

Günther Jung, Mohamed Hassen Khalifa and Anton Rieker Institut für Organische Chemie der Universität Tübingen, D-7400 Tübingen, Germany

Introduction of protecting groups The electrochemical oxidation of sterically hindered 2,6-ditert-butyl-4-aryl (or alkyl)-phenols U/2) yields phenoxenium cations These aryloxenium ions are formed via a homogeneous redox reaction between the intermediate cation radical 2 and the neutral radical 3, respectively via a two-electron process including the deprotonation of cation 2. Nucleophiles (NuH) react with the phenoxenium cation £ yielding para-substituted quinol derivatives ^ (R=aryl or alkyl).

0

R

Nu

5

0

R 4

C h e m i s t r y of P e p t i d e s a n d P r o t e i n s , V o l . 1 © 1982 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

194

This reaction can be carried out in neutral electrolyte systems in the presence of amino acid derivatives as nucleophiles yielding either N—protected or C—protected intermediates useful for peptide synthesis (3,4,5). a) Amino acid esters as nucleophiles The anodic oxidation of 2,6-di-tert-butyl-4-phenyl-phenol (3,5-di-tert^butyl-4-biphenylol) (1,2) in the presence of amino acid esters in dichloromethane followed by a saponification step gives N- [3 ,5-di-tert-butyl-4-oxo-1-phenyl-2,5-cyclohexadienyl] amino acids 7 (R3=H) in 80-95% preparative yields (3,4) .

'6 5

£

1

As shown on more than 10 different derivatives synthesized so far N-PChd amino acids are readily crystallizable compounds free of racemate. b) N-protected amino acids as nucleophiles Another application of the anodic oxidation of sterically hindered phenols is the preparation of quinol esters 8 from 2,6-di-tert-butyl-4-phenyl-phenol 6 and Z-resp.Boc-amino acids as nucleophiles. Quinol esters, e.g. Z-amino acid-^3,5di-tert-butyl-4-oxo-1-phenyl-1,5-cyclohexadienyl^ esters 8 corresponding peptide derivatives

or

constitute a new class of

carboxy protected intermediates. Via CF^COOH catalysis cleaving one of the tert-butyl groups they can be transformed into

the ether soluble pyrocatechol esters 9. These isolable

activated esters 9 couple to amino groups of amino acid and peptide esters often in more than 80 % yield.

195

6

• Z-NH-CR1R2-C00H -2e° • 2 H ® CH2CI2j (EtJ^NBF^

0

0

iH

0-fc-CR1R2-NH-Z

0 o-£-cr1r2-nh-z

cf3cooh^ch2ci2 -h

2

c=c(ch

3

)

2

Removal of N-PChd group The cleavage conditions for the N-PChd group are either mild acidolysis, e.g. 10-50 % CF 3 COOH in CH 2 C1 2 within about 15 min, or hydrogenolysis, e.g. H 2 /Pd-C (10 %) in CH 3 OH/HCl within

ca.1h. Therefore the PChd protection is selectively

applicable in combination with the Z, Bzl and the Boc, Bu*" groups.

• 2CFoC00H

2. resP-

ch2ci2

• CF3C00H x H2N-CR1R2-COOR3

N-PChd-Peptides H^/Pd-C

4

• hci x h 2 n - c r ' r 2 - c o o r ^

hci/ch3oh

The pyrocatechol trifluoroacetate J_0 is easily separated from the peptide trifluoroacetates by extraction with diethyl ether. In the same way the regenerated phenol 6 can be isolated from peptide hydrochlorides, and it is reusable. Recent experiments have shown the electrochemical reduction to be a third alternative way for deblocking N-PChd protected peptides. The NPChd group is completely stable towards the alkaline conditions used for saponification of peptide esters and the aminolytic conditions used for removal of the Fmoc group. Due to the simultaneous hydrogenolytic and acidolytic sensitivity of the PChd group and its electrochemical oxidative intro-

196

duction and reductive removal a variety of orthogonal combinations of protecting groups is possible in particular, when we have side chain protected PChd amino acids available. Furthermore, the different sensitivities of the PChd group can still be modified, e.g. by introducing suitable substituents on the phenyl ring. Moreover, by substituting the 4-phenyl for the 4-tert-butyl group on phenol 6 one can obtain N-(1,3,5tri-tert-butyl-4-oxo-2,5-cyclohexadienyl) amino acids, which can be transformed into further interesting derivatives (6). It is interesting that the rate of trifluoroacetolysis of N-PChd amino acids is much faster for «:,cC-dialkyl than for the protein amino acids. Thus, PChd-Aib-OH is cleaved quantitatively within 1 min and PChd-Ala(Gly or Leu)-OH within 5-10 min to about 70 % using 3 % trifluoroacetic acid in dichloromethane. Peptide synthesis with N-PChd protection The applicability of this new amino and carboxy protection and the acid -catalyzed carboxy activation is shown so far on the synthesis of a number of oligopeptides containing only difunctional residues. The extension of the new protective principle to combinations of protecting groups for the trifunctional amino acids is in progress. Representative examples of synthesis revealed readily crystallizable N-PChd oligopeptides with excellent solubilities in common organic solvents, complete retention of configuration and the possibility of monitoring reactions by UV detection. So far we prepared more than 20 N-PChd-protected oligopeptide esters, amides and acids, including 1 tetrapeptide, 1 hexapeptide and 1 heptapeptide. As a first example the wellknown C-terminal tripeptide of oxytocin, H-L-Pro-Leu-Gly-NI^ was synthesized. In a second test synthesis we built up by fragment condensation the lipophilic heptapeptide segment 14-20 of human lymphoblastoid interferon HuIFN-o£(Ly)

Ala-

Leu-Ile-Leu-Leu-Ala-G-ln.The same peptide had been prepared in our laboratory using Z- and Boc-protection.

197

H

N m s o

ñ I-i Ct!

H

N w

9

+J c a> e C P «j M

w (D S o M

(31 •H >1 O ÍN

W Q

l-l

-a ni tf o O O Ph FM Ph

I S fa H ¡E -P a a> e tn d)

CÖ H 1

a) h -p c 0) o a o o

6'¿-0-PM3d

a

0 " 0Jd l-O-PMOd

>i iH 0 1 3 O) J I

0 u cu 1 h-q i T3 XI U & m O N

.T-3-PMOd ,7-0-PM0d X-O-PWOd

.l-O-PMOd s'e-0-PM0d

a

UZl UZl 5821

a

8 a t i - i — 9'Z-O-WCtí — r m re7tJ —rrsvt L ¿ 671

o . = -9.65 ppm

(20 RSS) M-D-Ala-Phe-OMe (20 RRS)

6(CF3> = -10.1 ppm Scheme 4

References 1. 2. 3.

4.

5.

Urban, R., Marquarding, D,, Ugi, I.: Hoppe Seyler's Z. Physiol. Chem. ¿59, 1541 - 1552 (1978). Urban, R.: Tetrahedron 35, 1841 - 1843 (1979). Urban, R. (unpubl. results) see also: Urban, R., Marquarding, D., Ugi, I.: Proceedings of the American Peptide Symposium 1979. Ugi, I., Marquarding, D., Urban, R,, in: Chemistry and Biochemistry of Amino Acids, Peptides and Proteines. Vol. 6 (Weinstein, B., edit.). Marcel Dekker, New York (1981, in print). Brandt, J., Jochum, C,, Ugi, I,, Jochum, P.: Tetrahedron 33, 1353 - 1363 (1977).

208

6.

Dugundji, J., Kopp, R., Marquarding, D., Ugi, I.: Top. Cürr. Chem. 75, 1 65 - 1 80 (1 978) .

7.

Ugi, I., in: The Peptides. Vol. 2 (Meienhofer, J., edit.). Academic Press, New York 1979, p. 365 - 381.

8.

Marquarding, D., Burghard, H., Ugi, I., Urban, R., Klusacek, H.: J. Chem. Res. (S), 82 - 83 (1977); J. Chem. Res. (M), 091 5 - 0958 (1 977) .

9.

Eberle, G., Lagerlund, I., Ugi, I., Urban, R.: Tetrahedron ¿4 , 977 - 980 (1 978) .

10.

Herrmann, R., Ugi, I.: Tetrahedron 37, 1001 - 1009 (1981).

11.

Giesemann, G., v. Hinrichs, E., Ugi, I. (unpubl. results).

12.

Gieren, A., Dederer, B., George, G., Marquarding, D., Ugi, I.: Tetrahedron Lett., 1503 - 1506 (1977).

13. 14.

Falou, S., Ugi, I. (unpubl. results; see also ref. 4). Waki, M. , Meienhofer, J.: J. Amer. Chem. Soc. 9j), 6075 6082 (1977). Waki, M,, Minematsu, Y., Meienhofer, J., Izumiya, N.: Chem. Lett., 823 - 824 (1979),

15.

16.

(a) Bukall, P., Ugi, I.; Heterocycles (Sendai) 11, 467 470 (1978). (b) Bukall, P., Ugi, I.: Heterocycles (Sendai) 381 390 (1 981 ) . v. Zychlinski, Ugi, I,, Marquarding, D.: Angew. Chem. 86, 517 - 518 (1974); Angew. Chem. Internat. Ed. 13, 473 - 474 (1974).

17.

Bukall, P.: Dissertation, T. U. München (1980).

18.

Gil-Av, E., Charles, R., Bukall, P., Ugi, I. (unpubl. results) . Giesemann, G.: Dissertation, T. U. München (1981, in preparation) .

19. 20.

21. 22.

23.

Eckert, H., Breuer, W, , Geller, J., Lagerlund, I., Listl, M., Marquarding, D., Seidel, P., Stüber, S., Ugi, I., Zahr, S., v. Zychlinski, H.: Pure Appl. Chem. 51, 1219 - 1233 (1 979) . Sullivan, G. R., Dale, J. A., Mosher, H. S.: J. Org. Chem. ¿8, 2143 - 2147 (1973), Aigner, H., Koch, G., Marquarding, D,, in: Chemistry of Peptides and Proteins. Ill USSR - FRG Symposium. Makhachkala, October 2 - 6 , 1980. (Voelter, W. , Wünsch, E., Ovchinnikov, Yu. A., Ivanov, V. T., edit.). Walter de Gruyter, Berlin, New York 1981. Breuer, W.: Dissertation, T. U. München (1981, in preparation) .

ISOCYANIDES AS ACTIVATING REAGENTS IN PEPTIDE SYNTHESIS

Helmut Aigner, Gertraud Koch

and Dieter Marquarding

Organisch-Chemisches Institut der Technischen Universität München, D-8046 Garching, Germany

Dicyclohexylcarbodiimide

(DCCI) plays a major role as reagent

for a one-pot coupling of properly protected amino acids yielding peptide derivatives^. The wide application of this reagent is due to the fact that it is readily available and easy to apply. The real breakthrough in its use, however, originates from the discovery that racemization during the coupling in many cases can be totally suppressed, or at least remarkably decreased, if the carbodiimide is employed in combination with certain additives, such as hydroxysuccinimide (HOSu) or hydroxybenzotriazole (HOBt)1. The effectiveness of DCCI in amino acid coupling has been the impulse for numerous attempts to use in an analogous manner other compounds containing a similar "reactive moiety" as carbodiimides, namely the C=N double bond. These investigations have been undertaken not only to get informations about the behaviour of those compounds in condensation reactions, but also possibly to find a reagent which could replace DCCI, i. e. to find a coupling reagent that avoids the two major shortcomings of DCCI, namely its skin irritating property and the low solubility of its product dicyclohexylurea, which very often makes the purification of the desired peptide product so difficult. Up to now, however, in practice none of the compounds in the literature seems to be able to compete with DCCI. Attempts to introduce simple alkyl and aryl isocyanides as coupling reagents have not been satisfactory with respect to the

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter &. Co., Berlin • New York

210

2 3 4 yields of the desired peptides ' ' . The yields are particularly low, when as the acid component amino acids are used whose amino group is protected through a t-butoxycarbonyl or benzyloxycarbonyl group, i. e. those protecting groups which are usually employed in peptide chemistry"^. It is noteworthy, however, that after the normal work-up procedure the desired peptide derivatives usually are obtained in analytically pure form, even in those cases in which the yields are extremely low. When DCCI is used as coupling reagent, very often the yield of the condensation product is remarkably increased, if HOSu is 1

added to the reaction mixture . We have found that addition of HOSu, or HOBt, has almost no influence on the yields of the peptides, if simple alkyl or aryl isocyanides serve as condensation reagents. However, a large effect is observed, when isocyanides with tertiary amino groups are used. The best results have been obtained in the case of those isocyanides, in which the distance between the tertiary amino group and the isocyanide group is determined by an ethyl or propyl moiety. A very effective condensation reagent is N-(2-isocyanoethyl)-morpholine

(MAI) which has

not yet been described in the literature. It is prepared by wellknown procedures in two steps from the inexpensive chemical N-(2-aminoethyl)-morpholine. Most important with respect to the application of MAI is the fact that it does not have the customary smell of isocyanides. Preparation of MAI O

^

N-CH n -CH 0 -NH_ + HCOOH

/

z

Z

z

0

^

N-CHo-CH_-NH-CH0

^

¿.

Z

0.7 mole of N-(2-aminoethyl)-morpholine and 30 ml of formic acid in 300 ml of toluene are refluxed with a water separator. After the removal of the water the mixture is concentrated i. vac. and subsequently dissolved in dichloromethane. The solution is satu-

211

rated with ammonia (gas), filtered, and again evaporated i. vac.. The residue is distilled i. vac., N-(2-N1-formylaminoethyl)morpholine (b, p. 122 - 124°, 0.05 torr) is obtained in 90% yield. H-NMR (CDC13): -CHO 8.0 ppm (s) [TMS as internal standard] IR (neat): C=0

/ 0

1670 cm -1 .

\ N-CH-,—CH--NH-CHO + COCln 2

2

2

2

Et^N

- 2 HC1 - C0 2

•

/ \ O N-CH--CH..-NC 2 2 \_/ MAI

1 mole of N-(2-N1-formylaminoethyl)-morpholine, 320 ml of triethyl amine and 450 ml of dichloromethane are the starting materials. 1 mole of phosgene is introduced while stirring and cooling with ice. The mixture is allowed to reach room temperature and is stirred for 3 hours. Within 1 hour 40 g of ammonia (gas) are introduced (until saturation) while cooling with ice, and the mixture is filtered and concentrated. The residue is distilled i. vac., MAI (b. p. 70°, 6.05 torr) is obtained in 76% yield. IR (neat): isocyanide 2160 cm ^

From numerous experiments the following general procedure for the preparation of peptide derivatives has emerged. Procedure for preparing peptide derivatives The acid component (10 mmole);together with the additive, HOSu (20 mmole) or HOBt (10 mmole), is dissolved or suspended by stirring in a suitable solvent (dichloromethane, ethyl acetate, dioxane, isopropanol, t-butanol, tetrahydrofurane, or dimethyl formamide). After adding MAI (12 mmole) the reaction mixture is stirred for half an hour, then the amine component as hydrochloride (10 mmole) and a suitable tertiary amine (10 mmole) are added (triethyl amine, 4-dimethylaminopyridine (DMAP)).

212

In general, the reaction mixture is stirred for 24 hours and then after evaporation i. vac. the residue is distributed between ethyl acetate/1 N hydrochloric acid. The ethyl acetate phase is washed with 1 N hydrochloric acid, water, twice with 7.5% NaHCO^-solution, and once again with water. After drying over sodium sulfate the solution is evaporated i. vac. Note that it is necessary to let the acid component, the additive, and the isocyanide react over a suitable period of time before the amine component is added.

In Table 1 the yields of a number of peptide derivatives are given, which have been prepared according to the above procedure. It contains di-, tri-, and tetra-peptides. However, larger peptide segments have also been combined successfully^. Table 1: Peptide derivatives which have been synthesized according to the above general procedure. Peptide derivative

additive

base

solvent

Z-Val i Gly-OMe

HOSu

Et 3 N

AcOEt

81

Z-Val*Gly-OMe

HOBt

DMAP

t-BuOH

82

BOC-Aib^Aib-OMe

HOBt

DMAP

C

60

TCBOC-Val^Aib-OBzl

HOBt

TCBOC-Aib*Pro-OBzl

—

TCBOC-Aib*Ala-OBzl

HOSu

BOC-Gly-Ala-tVal-OtBu

HOSu

yield [%]

DMAP

6H6+) CH 2 CI 2

81

Et

CH 2 CI 2

69

N

3 DMAP —

CH 2 CI 2

80

CH 2 CI 2

95

BOC-Gly-Ala-^Leu-OEt

HOSu

Et^N

AcOEt

93

TCBOC-Aib*Val-Aib-OBzl

HOSu

DMAP

CH 2 CI 2

64

BOC-Gly-Leu±Ala-Gly-OEt

HOSu

Et 3 N

CH 2 CI 2

72

BOC-Gly-Ala-tLeu—Gly-OEt

HOSu

Et 3 N

CH 2 CI 2

70

TCBOC-Aib-Ala*Aib-Ala-OBzl TCBOC-Aib-Pro*.Val-Aib-OBzl

HOBt

DMAP

CH 2 C1 2

55

HOSu

Et N

CH 2 C1 2

61

boiling after 3 h of reaction time

213

The critical issue in peptide synthesis is the racemization of the acid component during the coupling step. MAI as well as DCCI have been applied in the synthesis of TFA-Phe-Phe-OtBu from the racemization prone TFA-Phe-OH and H-Phe-OtBu. The racemization of the phenylalanine moieties has been measured according to the method developed by Gil-Av et al. 6 Preliminary results 7 are most promising (see Table 2). It is very surprising that the additise HOSu suppresses racemization only in those cases completely in which MAI is employed as coupling reagent. Table 2 : Racemization of TFA-Phe-OH [1 mmole] during the formation of TFA-PKe-Phe-OtBu. additive

coupling reagent

[mmole]

[mmole]

HOSu [2]

MAI [1.2]

HOSu [2]

MAI [1.2]

HOSu [2]

DCCI [1.1] MAI [1.2]

phenylalanine D and L [%] D 0 L 1 00 D 0 L 100 D 0 L 100 D 3 L 97 D L

remarks starting material H-Phe-OH

addition of a catalytical amount of DMAP

T7 83

If the condensation reaction with isocyanides is performed in absence of any additive, then the mechanism of the amide bond formation may be described by a scheme which is given below. The main step in this scheme is the a-addition of the acid component at a formally divalent isocyanide carbon. All of the consecutive steps represent nucleophilic substitutions, in which the various nucleophiles that are present in the reaction mixture attack the carbonyl carbon of the acid component. The ability of isocyanides to undergo a-additions is well-documented in the g

literature . The intermediate products VII and VIII have been detected; the diacylamine derivative IX, which originates from a

214

g Mumm rearrangement , has, however, not yet been found. Compound IX corresponds to the diacylurea derivative which is often a byproduct in condensation reactions performed with carbodiimides in the absence of HOSu. A nucleophilic attack at the carbon atom of the imine leads ultimately to compounds which already belong to the scheme. +r3-nh R -CO-O-CO-R

VII

IV

f

• •>

V

+ 1 -VI R^ —COOH + C N - R 2

II

Hv \

2

R 1 -CONH-R 3 + OCH-NH-R2

R —CO —0

VI

[R1 = -CH-NH-CO-R4] -VI

OCH-N-R

I

IX

CO-OR

N-CH-R 4 // \ R -C

+ IV

V

C=0 VIII

The presence of additives such as HOSu may change the reaction mechanism considerably, in particular, when MAI is the condensation reagent. We have observed that in a solution of 1.2 moles of MAI and 2 moles of HOSu in dichloromethane no isocyanide is detectable any more according to IR. This ratio depends strongly on the nature of the solvent as well as on the acidity of the additive. For example, in order to achieve this effect in DMF, a larger amount of HOSu must be added. In the case of HOBt, which is more acidic than HOSu, the corresponding ratio is given by 1,2 moles HOBt and 1 mole MAI in dichloromethane. A protonation of MAI according to the next scheme explains these observations. Isocyanides are known to form strong hydrogen bonds

In the case of unsubstituted isocyanides a very large

excess of HOSu is needed, in order to protonate the isocyanide

215

such that it cannot be detected in the infrared. Note that the propyl analog of MAI is also not as easy to protonate as MAI itself.

/

CH,-CH9

IN \

C=N-CH0-CH~-N 2 Z \

0

/

^

\^ .N

CI

®IÔ-Su —

H

I

\

0

V U /

CH_-CH. 2 / 2 \X

\

C—H-Furthermore, when an excess of MAI (or a basic solvent such as pyridine) is used in the reaction, then very often the HOSu or HOBt ester, resp., of the acid component can be isolated in high yields. It is noteworthy that this is possible even in the presence of the amine component! The formation of these so-called "active" esters can be rationalized according to the following reaction scheme. Here the hydroxysuccinimide anion serves as the nucleophile which reacts with the a-adduct of the isocyanide and the acid component.

C=N-CH2-CH2-N

(excess) + HOSu + R 1 -COOH (+

3

R -NH2)

,CH0-CH.

/

IN.

CI

CH--CH-

.AN H"

/ p

2

+ IN

\N

\ - H

® lÔ-Su

R 1 -C0-0

R -CO-OSu (+

+

/—\ 0CH-NH-CH_-CH 2 2o-N \ /0

RJ-NH2)

216

It has been assumed that in condensation reactions with DCCI the additive HOSu reacts with the DCCI-adduct of the acid component to form the HOSu ester of the latter. This reaction is supposed to take place before the DCCI-adduct can decompose into the racemization prone oxazolinone derivative of the acid component'' ^ . Our observations are evidence against such a reaction mechanism.

References 1.

Wünsch, E.(edit.): Synthese von Peptiden. Teil II. Methoden der organischen Chemie. (Müller, E., edit.). Band 15/2, Georg Thieme Verlag, Stuttgart 1974, p. 103 - 119.

2.

Yasuda, N., Ariyoshi, Y., Toi, K,: United States Patent, 3,933,783. Jan. 20, 1976.

3.

Aigner, H., Marquarding, D.: Tetrahedron Lett., 3325 - 3326 (1 978) .

4.

Wackerle, L.: Synthesis, 197 - 198 (1979).

5.

Wünsch, E., Moroder, L, et al, (unpubl, results).

6.

Gil-Av, E., Feibush, B,, Charles-Sigler, R., in: Gas Chromatography. (Littlewood, A. B., edit.). Institute of Petroleum, London 1967, p. 227. Gil-Av, E., Feibush, B., Charles-Sigler, R,: Tetrahedron Lett., 1009 - 1015 (1966).

7.

Charles, R., Gil-Av, E., Aigner, H., Koch, G., Marquarding, D. (unpubl. results).

8.

Ugi, I. (edit.): Isonitrile Chemistry. Academic Press, New York and London 1971, p. 65 - 92,

9.

Mumm, 0., Hesse, H., Volquartz, H.: Chem. Ber. 48, 379 391 (1915).

10.

Schleyer, P. von R., Allerhand, A,: J, Amer. Chem. Soc. 84, 1 322 - 1 323 (1 962) . Ferstandig, L. L.: J. Amer. Chem. Soc. 84, 1323 - 1324 (1962). Ferstandig, L. L.: J. Amer. Chem. Soc, 8£, 3553 - 3557 (1962) . Allerhand, A., Schleyer, P. von R.: J, Amer, Chem. Soc. 85, 866 - 870 (1965).

11.

Rich, D. H., Singh, J,, in: The Peptides. Analysis, Synthesis, Biology. (Gross, E., Meienhofer, J,, edit.). Vol, 1. Academic Press, New York 1979, p, 249.

IMPACT

OF

CONFORMATION

ON

THE

SYNTHETIC

STRATEGIES

FOR

PEPTIDE

SEQUENCES

Manfred Mutter, Hermann Anzinger, V . N . R a j a s e k h a r a n P i l l a i (+) Institute of Organic C h e m i s t r y , D-6500 Mainz, Germany

Karsten

Bode,

University

of

Franz

M a s e r and

Mainz,

Introduction

The

physicochemical

rate

of

wise

and

coupling segment

peptides.

The

influence

upon

wise are

the

synthesis.

In

sequence,

and

physical of

the

length

of

the

peptide

and

coupling

a

growing

nature

of

environment concerned

peptide

by

the

decide

interdependence

of

properties

peptide

and

protein

we

sequences.

solubility

On leave Calicut,

of

chain

observed

a

their

to

kinetics

pre-

knowledge

of

influence

on

would for

permit

the

desired these

with

between

con-

the

and

respect on

of

The

groups

solvation

Chemistry,

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter &. Co., Berlin • New York

properties

delineate

correlation

f r o m t h e D e p a r t m e n t of Kerala-67635, India

step-

oligopeptides

and

of

transitions.

preferences

coupling

during

conformational

model

the step-

significant

these

peptides

length

and the

synthesis

rates

strategies

of

in

protecting

and

In o r d e r

a number

and

has

thorough

the

conformational

The

the A

factors

synthetic

sequence,

structure.

formation,

(+) of

their

composition,

condary

of

synthesized

investigated to

planning

role the

chain,

involved

peptides.

these

solubility

conformational

physicochemical

aspects,

the

solubility

the

effective

as

important

approaches for

the

an

such

extremely

influenced

primary the

an

condensation

chain

drastically

ferences

properties

play

se-

growing

University

218 peptide

chain

specific ful

synthesis the

of

proved

be h i g h l y

to

The

analysis

peptides

peptides

ficant This

high

Chain

on

of

characterized and

suitable

using

experimental

influence

property

out

could

since

the the

POE

CD,

Elongation

and

been

its

success-

phase

conformational

under for

the

applied

conformational to t h e

to h a v e

of the

bound

structure

on

different

conformational

coil

a

NMR m e a s u r e m e n t s

shown

conformations to

liquid

the

IR a n d

peptides

has

for

the

peptides. the

techniques

POE

is d u e

to d e l i n e a t e followed

be d i r e c t l y

by a s t a t i s t i c a l

segmental

be

oligopeptides

(POE)-bound

conditions. of

the

carried

to

to m e d i u m - s i z e d

of

was

helped

strategies

short

polyoxyethylene

bound

studies

synthesis

analysis the

these

experimental

For

method^

from

POE-

no

siqni? peptides .

behaviour

with

low

density

mobility.

and

Physicochemical

Properties

of

Homo-

oli g o p e p t i d e s The of

stepwise

synthesis

homooligopeptides

oxyethylene,

reveals

transitions

of the

properties.

The

of the

and

on

the

growing

amino

group

oligopeptides

exhibiting

homooligomers

of

aggregate drastic

at

example,

when

derivatives of

10%

the

very

decrease

valine short

viscosity

like

structure

ween

the

is

peptide

on

lengths with

viscous

chains.

by

Thus the

and

the

reduced

the

to

from

peptide at

in

POE-bound

tendency

the

chain.

For

tetrapeptide

concentrations

concentrations

polar

reactivity

in the

chloride

{ aggregated

be a g e n e r a l

certain

this

n = 6-8.

of

solvents change

development

by c o n f o r m a t i o n a l

conformational

1. The o c c u r e n c e

peptides

in

conformation

o f the

of

peptide

n >12,

that

(n 5)

in

these

behaviour

in t h i s

lengths),

synthesis.

result

due

. Then,

of

(5-conformation

insertion

segments

segments

'

this

of a 13-structure,

peptide

preferences

longer

a minimum

tendency

and

than

must

chain.

be e n v i s i o n e d

Pg-potentials

sequence

chain

by

various possible 9 10

to a v o i d

with

of

product

peptide

conformational

other

present

show

the

described

be o f c o n s i d e r a b l e

chains

aspects,an

to

be

solubility.

of the

is p o s s i b l e ,

the

examples

formation

f o r (3-structure of

peptides

is r e l a t e d

to

to

in

elongation

strategies

larger

by a d e c r e a s e

further

of m e d i u m - s i z e d

middle

chain

structures

densation

it

The

synthesis

ventional

Whenever

of peptides.

in p e p t i d e

at

help

strategies

of a p e p t i d e

peptide

can

cooligopeptides

experimental

of

conformational

stage

condensation,

prevents

These

and

interdependence and

(3-1 i ke s t r u c t u r e s

(a)

where

on the

synthesis

The

critical

either

more

form

Peptides

elongation

paralleled

general.

in c a s e s

the

that medium-sized

this

segment

the

reactivity

segments

show

by

be

observation

solubility short

extent,

for

properties

of h o m o o l i g o p e p t i d e s to

suggest

appears

investigations

physicochemical

not necessarily The

Strategies

pre a way

residues and when

in a

the

central

227 position these the

of

total

which ions

the

segments

should

the

sequence

gives and

concerned by

due

the

help

synthesis,

by

segment

reactivity

chain

1ength.

the of

to the

two

low

the

to the

and

main

Such

a

properties

obstacles

solubility

terminal

synthesis

finally

of

amino

in

of

establish procedure

conformational

physicochemical

to o v e r c o m e namely

proceed

approach

condensation.

consideration

dependent

duced

segment;

stepwise

transit-

of

peptides

peptide

segments groups

and

with

the

re-

increasing

Acknowledgement We

thank

the

support

of

"Chemie

und

Deutsche

this

work

Physik

Forschungsgemeinschaft through

der

the

for

the

financial

Sonderforschungsbereich

41,

Makromoleküle".

References 1.

M u t t e r , M . , B a y e r , E.: in " T h e P e p t i d e s : A n a l y s i s , S y n t h e sis a n d B i o l o g y " , V o l . II, G r o s s , E . , Me i en ho f e r , J.: Eds., A c a d e m i c P r e s s , N e w Y o r k , 1 9 8 0 , p. 286

2.

Mutter,

3.

M u t t e r , M., M u t t e r , H., m e r s , 15^, 917 ( 1976 ).

Uhmann,

4.

Rahman, S.A., 173 ( 1 9 8 0 ) .

H.,

5.

von D r e e l e , P . H . , L o t a n , Andreatta, R.H., Poland, c u l e s 4 , 4 0 8 ( 1971 ) .

6.

Ribeiro, A., Goodman, 100, 3903 (1978).

7.

Toniolo, C., Bonora, G.M., M a k r o m o l . C h e m . , 1J52 , 1997

Mutter, M., ( 1981 ).

8.

Toniolo,

Mutter,

M.:

C.,

Macromolecules, ^0,

Anzinger,

Bonora,

1413 R.,

Mutter,

(1977). Bayer, M.:

E.:

Biopoly-

Biopolymers,

19,

N., A n a n t h a n a r a y a n a n , V.S., D., S c h e r a g a , H.A.: M a c r o m o l e -

M.,

G.M.,

Naider,

F.:

J. A m .

Chem.

Soc.,

Pillai,

V.N.R.:

M., Pillai,

V.N.R.:

228 Makromol. 9.

C h e m . , ¿ 8 2 , 2007

(1981).

T a n a k a , S., S c h e r a g a , H.A.: M a c r o m o l e c u ! e s , 9, 142

10. C h o u , P.Y. (1978).

F a s m a n , G.D.: Ann. Rev. B i o c h e m . , 47, —

(1976).

251

SYNTHETIC STUDIES OF NEUROTOXIN II FROM VENOM OF CENTRAL ASIAN COBRA Naja naja ox-Lana

Vladislav Deigin, Victor Ulyashin, Inessa Mikhaleva, Vadim Ivanov Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR

At the previous FRG-USSR Symposium and at the 15th European Peptide Symposium we reported some results of our investigation aimed at total synthesis of neurotoxin from the venom of Central Asian cobra Naja naja oxiana (Fig. 1) (1). Synthesis of neurotoxin II is a component part of the structure-functional investigation of polypeptide neurotoxins and the development of methodology for synthesis of large peptides and proteins. NT-II is a 61-membered polypeptide with 4 disulfide bridges.

Fig. 1. Amino acid sequence of neurotoxin II from the venom of Central Asian cobra Naja naja oxiana.

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter & Co., Berlin • New York

230 The synthesis was elaborated in such a manner so that we could employ the strategy of maximal protection of the trifunctional residues with the help of hydrophobic groupings of the benzyl type as was described in an earlier paper for the case of obungarotoxin

(2). In view of the fact that we encountered

some difficulties in synthesizing a-bungarotoxin, for instance, side reactions involving the liberating cysteine SH group due to HF treatment, we utilized the acetamidomethyl protection instead of

p-methoxybenzyl. Such a combination of the protec-

ting groups allows the purification of the resulting product following HF deblocking to be carried out without any risk of oxidation of the cysteine residues. The glutamine carboxamide groups

and tryptophan

indole remained unprotected. The fol-

lowing protections were used for the remaining amino acids: benzhydryl for asparagine,benzyl esters for aspartic and glutamic acids, benzyl ethers for serine and threonine, 2,6-dichlorobenzyl for tyrosine and the tosyl groups for the arginine guanidine group. Tos, Boc and Dnp groups were used to protect the histidine imidazole.

The a-amino group was protected either

by Boc- or Z-group. Stepwise synthesis was the method used to obtain all the short fragments corresponding to the total amino acid sequence of the NT-IX molecule (Fig. 2), namely 1-3, 4-8, 9-12, 13-15, 16-19, 20-26, 27-33, 34-47, 48-51 and 51- 61. Each fragment was synthesized in more than one way that allowed us to optimize the yield and facilitate the purification of the final product. The present paper is devoted to further studies on condensation of the fragments, as well as on methods of their-, purification. Several attempts have been made to purify the protected peptides by gel-filtration on soft strongly swelling sorbents, as for example, enzacryl K-2 (3), Sephadex LH-20 and LH-60 (4) or Bio-Beads Sxl (5) . However,under these

conditions

it appeared difficult to realize the separation of peptides as they differ in molecular mass by less than twofold. Besides, difficulties connected with their purification

increased

with a rise in the molecular mass of the peptides.

231

Acm

Bzh

Fig. 2. Molecule fragment scheme for neurotoxin II.

Moreover, we encountered other difficulties in the process of purification of the intermediate protected NT-II fragments, especially when using the conventional gel-filtration methods (1) .

Analytical and preparative size-exclusion HPLC methods were employed in the case of the protected hydrophobic peptides in organic solvents. But in order to optimize these procedures we had to investigate various types of gels - soft, semirigid and rigid. Among the investigated soft gels, Sephadex LH-20 is in virtue of its properties the most suitable for separation and analysis of synthetic protected peptides with a molecular mass below 2000 dalton (Fig. 3). The mechanical properties of Sephadex LH-20 favour a rise in the efficiency of the column and linear flow rate.

232

Fig. 3. Chromatogram of the protected peptides and naphthalene mixture: M M M.M. 1. Aoc-56-61-OBzl 1963 2. Aoc-58-61-OBzl 1265 3. Boc-60-61-0Bzl 768 4. Boc-Asn(Bzh)-OBzl 488 5. Naphtalene 130 8 x 600 mm column, Sephadex LH-20 gel, eluent DMF . Flow rate 0.4 ml/min, refractive index detector.

With the help of narrow fractions of sorbent particles (40 50 u)

we managed to obtain an efficient column operation

(about 8000 t.p. per meter). A more detailed investigation of the soft and semirigid gels was carried out in (6). We used silica gels of different types to separate and analyze peptides with a molecular mass above 2000 dalton. The application of rigid sorbents in size-exclusion chromatography has opened the way for new opportunities for performing separation and analysis of peptides. Thus, for example, the accessibility of silica gel with small-size particles makes it possible to obtain an extremely high efficient column (about 100000 t.p. per meter). Additionally, the mechanical properties allow

operating at high flow rates. One can select

silica gels for working in any molecular mass range. By modifying the silica gel surface one can obtain a sorbent suitable for the separation of substances of any class, including labile

233

Fig. 4. Calibration curves for the studied sorbents with synthetic protected peptides in DMF. 1. o 2 . -4 3. £ 4.—A—

Sephadex LH-20 Silica gel "L" Silasorb 600 Spherical silica gel

5.—« 6. • 7. •

Si-40 PSM-60S Zorbax Sil

Synthetic peptides indicated in Fig. 4 caption as well as a Boc-52-61-OBzl fragment (M.M.=2575) and naphtalene were used for calibration. Void volume was determined using high molecular impurities.

biopolymers. Therefore,silica gels can be applied for analysis and separation in preparative quantities of practically any substance. With the help of different modifications it is also possible to prepare sorbents oh which the separation proceeds" according to a purely size-exclusion mechanism. We investigated the selectivity of different silica gels and the behaviour of peptides on them, with the aim to choose an optimal form for size-exclusion chromatography of protected peptides in organic solvents (Fig. 4). As is seen in Fig. 4 Zorbax Sil and PSM-60S have practically a linear calibration curve but their selectivity value is relatively low. Silica gel Si-40(Merck) also has the same linear calibration curve, although its selectivity is apparently less and the operating range narrower, most likely due to the larger

234 6

mln. 5

4

3

2

1 0

Fig. 5. Chromatogram of artificial peptide mixture:

M.M.

1. High molecular weight impurities . . 2. Boc-34-61-OBzl 5417 3. Boc-48-61'-OBzl 3125 4. Aoc-55-61-OBzl 1962 5. Aoc-58-61-OBzl 1265 6. Boc-60-61-0Bzl 768 7. Boc-Asn(Bzh)-OBzl 488 8. DMSO 78 8 x 250 mm column, Silasorb 600 sorbent, eluent DMF. Flow rate 2 ml/min, refractive index detector.

yolume of the micropores. The silica gel prepared according to Unger's methods (7) appeared to have the best selectivity, especially in the high molecular

weight region. The calibration curve of this sili-

ca gel has a non-linear shape. Besides, the nature of the silica gel surface also plays an essential role in achieving a suitable separation of the protected peptides.

For instance, only totally protected pep-

tides were eluted from the column with a non-modified surface by the pure size-exclusion mechanism (Fig. 5). Peptides containing free imidazole, guanidino or amino groups sorbed on a gel

235

1-12 13-19

20-26

mna .(H )»c« XCACBT XC/HOP*

27-33

3i-39 ¿0-47 ¿S-51 52-6i

B«c4 XL7I«

Fig. 6. Preparation scheme for fragments 1-33 and 34-61.

matrix and eluted a little later then the protected peptides of the same molecular mass. In the course of synthesis and condensation of the fragments we observed the formation of high molecular weight impurities

Fig. 7. Analytical (A) and preparative (B) chromatography of synthetic peptide of 34-61 sequence on a silica gel "L" column, 25 x 330 mm in DMF. Flow rate 10 ml/min. 1. High molecular impurities 2. Boc-34-61-OBzl 3. Initial fragments UV-detector,respect.refractive index detector. Loading under preparative conditions 50 mg per 20 ml.

236

of peptide nature whose amount increased with reaction time. Not always is it possible to successfully remove these polymer products by standard methods, especially on large peptides. According to our data these polymers are not products of peptide association, for their retention times do not change with rechromatography. The fragments were condensed mostly by DCC/HOBT method (Fig.6). Fragments 1-33 were obtained by the condensation of two blocks, that of 1-19 and 20-33. The C-terminal part of the molecule sequence was obtained by the same DCC/HOBT, as well as by DCC in the presence of pentafluorophenol. In both cases the yield varied within the range of 25-30%. The reaction product was separated by size-exclusion chromatography on a silica gel column. An example of the analytical (A) and preparative (B) separation of the condensation reaction products of fragments 34-37 and 48-61 is given in Fig. 7. The individuality of all the resulting fragments was also controlled chromatographically and by determining the N-terminal amino acids and was confirmed by amino acid analysis data. Thus, two large molecule fragments were obtained, those of 1-33 and 34-61 (Fig. 2).A search for optimal conditions for condensation of these blocks into a uniform polypeptide chain is still in progress.

References 1. 2.

3. 4.

Mikhaleva, I.I., Deigin, V.I., Ulyashin, V.V., Ivanov, V.T.: "Peptides 1978", Proceedings of the 15-th European Peptide Symposium, Poland, 391-395 , 1979 Ivanov, V.T., Mikhaleva, I.I., Volpina, O.M., Myagkova, M.A., Deigin, V.I.: "Peptides 1976" (ed. A.Loffet), Proceedings of the 14-th European Peptide Symposium, Belgium 219-229, 1976 Galpin, I.J., Handa, B.K., Kenner, G.W., Moore, S., Ramage, R.: J. Chromatogr. 123, 237-242 (1976). Danho, W., Fohles, T.: Hoppe-Seyler's Z. Physiol. Chem. 36, 839-847 (1980).

237

5. 6. 7.

Volpina, O.M., Deshko, T.N., Mikhaleva, I.I., Ivanov, V.T.: Bioorg. Khim. 6, 1155-1162 (1980). Deigin, V.l., Ulyashin, V.V., Nefedev, P.P., Zhmakina, T.P., Belen'ky, B.G., Ivanov, V.T.: Bioorg. Khim. (USSR) 8, in press Unger, K., Check-Kalb, J., Straube, B.: J. Polym. Col. Sci. 253, 658 (1974) .

I N V E S T I G A T I O N O N T H E S Y N T H E S I S O F S A L M O N C A L C I T O N I N II F R A G MENTS Gennadij Vlasov, Valentina Glushenkova, Valerij Lashkov, talija Kozhevnikova, Lidija Nadezhdina, Leonid Irina Ditkovskaja, Oskana Glinskaja and Tatjana

Na-

Krasnikov, Komogorova

I n s t i t u t e of M a c r o m o l e c u l a r C o m p o u n d s of the A c a d e m y of ces of the U S S R , L e n i n g r a d , U S S R

Scien-

Introduction S y n t h e s i s of the s a l m o n c a l c i t o n i n II f r a g m e n t s w a s

investi-

g a t e d u s i n g a c l a s s i c a p p r o a c h . A n u m b e r of b y - r e a c t i o n s the c o u r s e of the s y n t h e s i s w a s

in

investigated.

Results C a l c i t o n i n s are 3 2 - m e m b e r e d p o l y p e p t i d e h o r m o n e s w i t h a d i s u l f i d e b o n d b e t w e e n the f i r s t a n d the s e v e n t h a m i n o a c i d

(1).

T h e y r e g u l a t e the c a l c i u m a n d p h o s p h o r u s l e v e l s in the b l o o d (2). S a l m o n c a l c i t o n i n is m o r e a c t i v e t h a n o t h e r h o r m o n e s c e p t eal

calcitonin

ex-

(3).

In o r d e r to d e v e l o p a s a l m o n c a l c i t o n i n s y n t h e s i s the

in-

v e s t i g a t i o n of the s y n t h e s i s of its f r a g m e n t s w a s c a r r i e d

out

in s o l u t i o n by u s i n g a m i n i m u m a m o u n t of b l o c k i n g g r o u p s .

The

fragments

1-5, 6-10, 11-15, 16-21, 22-24,

s y n t h e s i z e d a c c o r d i n g to s c h e m e s

25-28 a n d 29-32 w e r e

1-7.

T h e s y n t h e s i s of f r a g m e n t 1-5 w a s c a r r i e d o u t by the use of the N P S b l o c k i n g g r o u p for the a m i n o f u n c t i o n s a n d D C C a n d (scheme 1). The s y n t h e s i s of f r a g 8 9 m e n t 6-10 w a s c a r r i e d o u t w i t h N P S (Leu , Val ) a n d Boc PFP m e t h o d s for c o u p l i n g

(AcmCys^, Thr^)

b l o c k i n g g r o u p s a n d D C C as the c o u p l i n g

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter &. Co., Berlin • N e w York

rea-

240

gent (scheme 2), Scheme 1 Cys1

Asn"

Ser

NPS-

Leu NPS-

-OMe

NPS-

-OMe

H-

-OMe

PFP

-OMe

NPSNPSAcm Boow^-OPFP

-OPFP

Ser"

H-

-OMe

NPS-

-OMe

H-

-OMe

Boc- ,Acm

-OMe

Boc- ,Acm

-NHNH,

Scheme 2 Thr

Cys

Boo BocH-

-OPFP

Val

-OH

^Acm —OPFP

H-

Leu'

Gly

10

Z- -OH

-OMe

Z-

-OMe

H-

-OMe -OMe

H ,Acm

-OMe

-Acm

-OMe

The pentapeptide 11-15 was synthesized from two fragments: 11-13 and 14-15. The carbobenzoxy blocking group was used for the protection of the c< -amino function and the tertbutyloxycarbonyl group for blocking the

¿-amino function

241

of lysine^. Condensation of fragments was carried out by Rudinger's modification of the azide method (scheme 3). The hexapeptide 16-21 was synthesized from two fragments (16-17 and 18-21) by the DCC/HOSu coupling method. The dipeptide 16-17 was prepared by the reaction of the N-hydroxysuccinimide ester of Z-leucine with histidine. The tetrapeptide 18-21 was synthesized according to the scheme 4. The carbobenzoxy and tert18

butyloxycarbonyl blocking groups were used for lysine The condensation of the acylamino acids and peptide fragments was carried out with the PFP, NP ester and DCC/HOSu method. Scheme 3 Lys 11

12 Leu NPS-j-OH

Boc Z — hQH

NPSH-

^Boc ffoc

Ser 13 -OMe

Gin 14 -OPFP

Asp 1' 5 OBut H— ^-OH ,

-OMe -OMe -OMe -NHNH, -N-

,Boc

The synthesis of the fragment 22-24 (scheme 5) was carried 24 out without blocking the guanidino group of arginine with the help of the activated ester method. The tetrapeptide 25-28 was prepared according to scheme 6 from the azide of the tripeptide 25-27 and glycine. The tetrapeptide 29-32 was synthesized according to scheme 7 from two dipeptides by the DCC/PFP method. Crystallization, gel chromatography and adsorption chromatography were used for the preparation of the calcitonin fragments in pure form. Many by-reactions were investigated. Dipeptide 31-32 can be prepared only with the use of prolin-

242

Scheme 4. Leu

16

17

His

Leu

Lys

19

Gin

20

21

Z- 3Np Z-OPFP

H-

Boc Z—-OH -OSu

H OH

^Boc J3oc

Scheme 5. Phe 22 -OSu

Pro

23

Arg 24

-OH "OH -OSu

-OH -OH

amide but not from the methyl ester. All attempts to transform the carbobenzoxy dipeptide ester into the amide were unsuccessful. The reaction of acylation of the methyl ester 25 of threonine or the methyl ester of threonyl-asparaginylthreonine (25-27) by acyl-(Z or Boc)nitroarginine with the help of DCC yield only N-acyl-urea (70-80 %). Only the 2.5-diketo-piperazine of valine and proline was found during the catalytic hydrogénation of carbobenzoxyvalyl-prolinamide in the absence of strong acid. However, in the presence of p-toluenesulfonic acid hydrogénation yields the dipeptide amide.

243 S c h e m e 6. Thr

25

Asn Z-

-OPCP

26 DNp

Thr

27

Gly

-OMe

Z-

-OMe

H-

-OMe -OMe -NHNH, -NH-

-OH -OH

S c h e m e 7. Ala

29

Gly

30

Z -+OSU

-OH

z-

-OH

A t t e m p t s to s y n t h e s i z e m o r e c o m p l e x p e p t i d e f r a g m e n t s w e r e m a d e a n d the d e c a p e p t i d e peptide

1 —10, p e n t a d e c a p e p t i d e

25-32 a n d h e p t a d e c a p e p t i d e

16-32 w e r e

1-15, octa-

prepared.

O p t i m a l c o n d i t i o n s of c o u p l i n g b u l k y f r a g m e n t s to ^he p e p t i de of the s a l m o n c a l c i t o n i n s e q u e n c e a r e u n d e r tion .

investiga-

244 References 1. Niall, H.D., Keutmann, H.T., Copp, D.H., Potts, Y,T.:Proc. Natl.Acad.Sei.64, 771 (1969), 2. Riniker, B., Reher, R., Maier, R., Byfield, P.G.H., Gudmundsson, T.V., Maclntyre, J.: Nature 220, 984 (1968). 3. Keutmann, H.T., Passons, J.A., Potts, J.T.: J.Biol.Chem. 245, 1491 (1970).

TOTAL SYNTHESIS OF SOMATOSTATIN WITHOUT HYDROXYL GROUP PROTECTION OF HYDROXYL AMINO ACID RESIDUES

Yu.P.Shvachkin, S.K.Girin, A.P.Smirnova, A.A.Shishkina, N.M. Ermak Institute of Experimental Endocrinology and Hormone Chemistry, Academy of Medical Sciences of the USSR, Moscow

Introduction The hormone somatostatin is a tetradecapeptide of structure (I) (all asymmetric amino acids have L-configuration) (1). 1 5 10 14 H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH I

I (I)

At present this hormone is of interest for peptide chemists as well as for clinicians. In spite of a number of published papers concerning the synthesis of somatostatin in solution and on polymeric carriers the problem of preparative methods for somatostatin remains nevertheless an actual one.

Results In pursuit of a research study on somatostatin derivatives we have performed a total chemical synthesis of this hormone according to a new scheme whose specific features are: 1) The possibility to use amino acid derivatives (Thr and Ser ) without hydroxyl group protection;

Thr

2) the application of the tetrahydropyranyl protection for masking mercapto groups in the Cys"^ and Cys ^ residues at intermediate stages of the synthesis.

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter &. Co., Berlin • New York

246

Preparation of the protected linear tetradecapeptide was performed by a fragment condensation starting from the previously synthesized 1-7 and 8-14 fragments. From the point of view of the tactics of the peptide synthesis this variant which makes allowance for "a docking" of the mentioned heptapeptide fragments is the most preferable one since in this case properties of the final desirable product differ maximally from those of the initial materials. This would facilitate the separation of the conceived tetradecapeptide from the reaction mixture. All condensation reactions were carried out under conditions which reduced racemization of the individual amino acid residues to a minimum. The key stages of the fragment docking were performed by the azide method or by the method of activated (pentafluorphenyl) esters. The tert-butyloxycarbonyl group or the o-nitrophenylsulfenyl group were used to protect the o-OSUp3] . H-1>«BOO-OKU] Z-Aij^LysBOa-OH^-U^ jtywci Z-G>uPeuhOSU [ l j •

H-AnlHCH-LyHBOC)-OH|a-Klj

Z-GMCI8u)-Afg(HQ)-Lys(B0C)-0H[l2-U^

1

'

2-ATA-QSU[9] . H-Pto-OH[Kg

Z-*t»IZj-OSU[l j

Z-Alo-Pre-OH [ 9 - I O j

.

H

mjluCCul-AtglHCIHyiaOCHOH[tt-Hlj

Z-Arg(Z2l-Glu(OISu)-Arg-LyslSOCI-OH§l-U^

IDCC/NOIU

j^Mff*

Z-Ala-Pnt-OSUfo-lOlj

.

K-Alj(Har)-OUl(Oeu>-Arg|Hei)- L»»(BOCI-OH [ l t - U ^

Z-Alo-Pio-Aig(HBt)-GlulOeu)-ArglHB>)-LyslBOC)-OH[9-U^

Ln

NPS-M»I-CHP[»]

.

H-AlQ-Pro-Af9lHBf)-Gtu(01Bu)-AnlHBfHy»lB0C)-0H[9-Ub|

N P S - M t t - A l a - P R O - A r g ( H B f ) - G I I I O ® u ) - A f g IHBrl-LyslBOC) -OH [ i - U o ]

Scheme 4. Synthetic route to fragment III corresponding to sequence 8-14 of somatostatin-28; for analytical data see Ref. 14; overall yield calcd. from Z-Arg(Z 2 >-OSU [ 13]: 29%. Z-Pro-05u[6] •

H-Alo-OCu[7]

Z - P r o - A t a - O f i u fc-la]

Z-Asn-0WP[5] • H-Pro-Aln-OIBu fS-7b]

Z - A s n - P r o - A l o - O C u ¡S-Tqj MJ/IW

H-Asn-Pro-Alo-OtBu [ 5 - 7 ^

|tfa

Z-S«flBu>-OSU[NH't>Cs+>Rb+>K+>Na+>Li+. Of considerable interest are the mean life-time and conductance values of these channels. As seen in Table 3 all bis-gramicidins,regardless of their bridge position and spatial structure form channels with similar conductance but with the mean life-time by 1-2 orders of magnitude longer than with the channels formed by gramicidin A. In addition to the long-living channels the head-to-tail and tail-to-tail analogs also form short-living channels. It are these channels which are responsible for the observed conductance in the noise experiments. Bearing in mind the aggregation tendency of these analogs revealed by spectral measurements in solution, and their high concentrations at noise experiments we assume that the shortliving channels are formed by bis-gramicidin associates (possibly, by + + TTTT double helices) . The head-to-head derivatives do not seem to form short-living channels at the great variety of membrane modifications. So, the covalent crosslinking applied to gramicidin A in this work failed to produce a conformationally homogenous species. Nonetheless the data obtained clearly indicate that the channel-forming capacity in the gramicidin series should not be considered as a prerogative of a single molecular structure. In particular, a whole family of ^D^LD' ^LD^LD anc^ ^LD^LD helices are efficient channel formers. As for the double helices, their channel forming capacity still requires further study.

267

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Veatch, W.R., Blout, E.R.: Biochemistry 13, 5257-5264 (1974). Urry, D.W.: Proc. Nat. Acad. Sei. USA 68, 672-676 (1971). Urry, D.W., Goodall, M.C., Glickson, J.D., Mayers, D.F.: Proc. Nat. Acad. Sei. USA 68, 1907-1911 (1971). Ramachandran, G.N., Chandrasekaran, R.: "Progress in Peptide Research" (S. Lande, ed.), Gordon and Breach, New York, p. 195 (1972). Ramachandran, G.N., Chandrasekaran, R.: Ind. J. Biochem. Biophys. 9, 1-11 (1972). Veatch, W.R., Fossel, E.T., Blout, E.R.: Biochemistry 13, 5249-5256 (1974). Shepel," E.N., Jordanov, St., Ryabova, I.D., Miroshnikov, A.I., Ivanov, V.T., Ovchinnikov, Yu.A.: Bioorg. Khim. 2, 581-593 (1976). Ovchinnikov, Yu.A., Ivanov, V.T.: "Biochemistry of Membrane Transport" FEBS-Symp. No. 42 (G. Semenza, E. Carafoli, eds) Springer-Verlag, Berlin, pp. 123-146, 1977 Sychev, S.V., Nevskaya, N.A., Jordanov, St., Shepel, E.N., Miroshnikov, A.I., Ivanov, V.T.: Bioorg. Chem. 121-151 (1980).

CYCLIC ANALOGUES OF BRADYKININ AND KALLIDIN

Gunars Chipens, Felikss Mutulis, Natalia Mishlyakova Division of Peptide and Protein Bioregulators, Institute of Organic Synthesis, Latvian SSR Academy of Sciences, 226006, Riga, USSR

Introduction Total semi-empirical space structure calculations for bradykinin have shown that energetically preferable are the bent, quasi-cyclic structures (1) characterized by ionic bonding between the guanidyl group of arginine in position 1 within the common fragment (2) and the carboxyl group of arginine in position 9. Experimental evidence in favour of the existence of bent structures in aqueous solution was obtained by Ovchinnikov, Ivanov and co-workers investigating labelled analogues of bradykinin by EPR and fluorescence spectroscopy (3,4). However, further analysis of NMR spectra of bradykinin (5) and titration data (6) have shown that there is no ionic bonding between the terminal arginine residues of the bradykinin molecule in aqueous solution. In our view, this fact can be easily explained by the existence of a hydration layer, screening the electromagnetic fields of ions. However, quasi-cyclic structures are possibly formed in apolar biophase during hormone-receptor interaction after the dehydratation of ionogenic groups. This assumption is supported by the fact that dehydratation of the contacting surfaces is the first step during substrate-enzyme interaction(7).

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter &. Co., Berlin • New York

270

u • H *

CM*

o a n o •P -H O -P •H U S H)

KO

CO » N (N m •

a)

a)

•H -P Ü

G

•H

O

_ O CM M o 10* M C 0) o H -H ft-p a) o a i rH O 1J

> •H -P O a) m m

en

,—.

j - i Ol M

rn

oo O

\

\

(U > •H -P O i H O (M O M CM CO >1-1

en

o>

a &
i rH U CM O M CM Cn M
1.


i -a «o m a

u >. o

-H o >1 rH Ol

r—i I rH

11)

G

>1

•H CO >1 1 (J

r

o -a o G -H

C •»H -a

(0 ¥ u I O rH U

>1

O

I

I 3 —

O

a

> —

C "ri C l-rl

i A: O >1 rH -O U (0 (H Ü

>1

-Ü (U N ~ • H CI) - P a) ai - P 3 •P fi M -H

>1.«

O c 10 o a)

S3

(0 o ai -a o T> 0 a •h

>1 n (0 n jq i ~ H >i o c

A

a) (0

(0 - H

G

1 3

(U -a o i—iiq

-P U w > o MH o ~

KAUC14,

K2PT(N02C1)2,

PB(CH3COO)2,

and

HgCl 2 as well as measurement and processing of experimental data were described in detail elsewhere (3, 4). The total numo ber of reflections from the 2.5 A range for each derivative used in the calculations was 3,43 3. The heavy atom binding sites were located by X-ray "direct" methods

(5). The selec-

tion of correct enantiomorph has been done using anomalous scattering data. The mean figure of merit < m > after last cycle of phase refinement was 0.80.

C h e m i s t r y of P e p t i d e s a n d P r o t e i n s , V o l . 1 © 1 9 8 2 b y W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

430 Results The actinoxanthin backbone folding drawn from C a atom coordinates is shown in Fig. 1. A distinctive feature of the actinoxanthin structure is the absence of a-helices and the presence of enhanced content of antiparallel B-structure (-55%). The three-dimensional architecture of the molecule has a double part appearance with the two lobes of different sizes (70 and 30%) being separated by a well-defined cavity. The larger lobe is a regular g-supersecondary structure containing the N- and C-termini.

Fig. 1. Topology of actinoxanthin spatial folding. The arrows indicate 6-strands, the strings indicate non-regular protein parts.

431 It is a slightly flattened cylinder formed by two layers of antiparallel g-sheets. The first external layer consists of three strands S^, S^ and S,- and the second one, bounding the molecular cavity, consists of four strands S^, S^, Sg and S^. g-Strands arrangement in protein architecture has topology of so called "Greek key" (6). The g-sheets exhibit a right twist when viewed along, and a left twist when viewed across the sheet. The two neighboring g-strands form an angle of 15-20° when looking across the sheet. The fragments of sharp chain reversal (g-turns) positioned between the g-strands are rich in Ser, Ala and Gly residues. The inner part of the g-cylindrical barrel is tightly packed with hydrophobic side chains oriented towards the axis: Ala(92), Val (6, 18, 20, 64), Phe(4, 51, 61, 63, 106), Leu(90, 99, 104), Ile(31); the Gln(33) side chain is also inside the cylinder. Hydrophilic side chains are arranged mainly on the external surface; the largest group among them is formed of Ser and Thr residues, which are located along the chain alternately with hydrophobic residues. They mainly populate the external threestrands g-sheet S, S3 c and strand S„. L ,A 4 The cylinder is bounded from one end by turns S2-S.J, S^-S^, Sg-S^ and the N-terminal and on the other end by turn disulfide bond Cys (83)-Cys (-88) , residue Lys(66) and the C-terminal. The smaller and less regular lobe of the molecule includes two closely spaced deca- and hexapeptide cycles 34-43 and 83-88 closed by disulfide bonds, curved fragment 43-47 and a g-hairpin shaped part 66-82 on the elongated loop between g-strands S r and S,. d o The internal area of the molecular cavity separating the two structural units contains a large number of Gly and Ala residues and disulfide bond Cys(34)-Cys(43). The peripheral part of the cavity is lined by the side chains of Tyr(30), Thr(49, 72), Asn(89 , 94 ), Gln(71), Ser (95) and His(101). The relative arrangement of the seven g-strands of the super-

432 secondary actinoxanthin structure shows strong topological similarity with the arrangement of g-strands in C and V domains of immunoglobulins IgG (7), in Bence Jones protein REI (8), in superoxide dismutase subunits (9), and in azurin (10). Despite the fact that these protein units consist of about the same number of residues and show close resemblance of the spatial folding pattern, the proteins themselves are functionally unrelated and have no evident sequence homology. Estimation of topological similarity of the actinoxanthin cylindrical subunit with those of immunoglobulin and superoxide dismutase has been performed on a computer by least squares method superimposing of their C a -frames. Pairwise comparison of the three-dimensional structures shows that the arrangement of practically half of amino acid residues in actinoxanthin and in the two other proteins are topologically equivalent; the root mean-square distances between the residues o were less than 2.5 A. The mean value of base change per codon for comparable protein ranges was about 1.31. It is unclear whether the observed structural similarity is indicative of divergent evolution of these proteins from a common ancestor or convergent evolution of independent precursors to energetically favorable topological state. The characteristic cavity found in the actinoxanthin structure undoubtedly has a direct relation to its biological activity. It is the probahle binding site for the chromophore in the active complex.

References 1.

Khokhlov, A.S., Cherches, B.Z., Reshetov, P.D., Smirnova, G.M., Sorokina, I.B., Koloditskaya, T.A., Smirnov, V.V. , Navashin, S.M., Fomina, J.P.: J. Antibiotics 22, 541-544 (1969) .

2.

Khokhlov, A.S., Cherches, B.Z., Reshetov, P.D., Smirnova, G.M., Koloditskaya, T.A., Sorokina, I.B., Prokoptseva, T.A., Ryabova, I.D., Smirnov, V.V., Navashin, S.M., Fomina, I.P.: Izvestiya AN SSSR (ser. biol.) (Bull. Acad. Aci. USSR,

433 Russ. ser. biol.) 755-763 (1970). 3. 4. 5. 6. 7. 8. 9.

Pletnev, V.Z., Kuzin, A.P., Trakhanov, S.D., Kostetsky, P.V., Popovich, V.A., Tsigannik, I.N.: Biopolymers 20, 679-694 (1981). Pletnev, V.Z., Trakhanov, S.D., Tsigannik, I.N.: Bioorgan. Khim. (Soviet J. Bioorgan. Chem) 5, 1605-1608 (19 79) . Pletnev, V.Z., Kuzin, A.P., Trakhanov, S.D., Popovich, V.A., Tsigannik, I.N.: Bioorgan. Khim. (Soviet J. Bioorgan. Chem.) 6, 563-569 (1980). Richardson, J.S.: Nature 268, 495-500 (1977). Amzel, L.M., Poljak, R.J.: Annu. Rev. Biochem. £8, 961-997 (1979) . E£>p, 0., Colman, P., Fehlhammer, H. , Bode, W. , Schiffer, M. , Huber, R.: Eur. J. Biochem. £5, 513-524 (1974). Richardson, J.S., Richardson, D.C., Thomas, K.A.: J. Mol. Biol. 102, 221-235 (1976).

10. Adman, E.T., Stenkamp, R.E., Sieker, L.C., Jensen, L.H.: J. Mol. Biol. 123, 35-47 (1978).

ALDIMINE BOND MIGRATION IN THE PHOTOCHEMICAL CYCLE OF BACTERIORHODOPSIN

Najmutin Abdulaev, Victor Tsetlin, Alexandr Kiselev, Valdis Zakis, Yuri Ovchinnikov Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences,Moscow, USSR

Introduction We found that a migration of the aldimine bond takes place in the course of the bacteriorhodopsin

(BR) photochemical

cycle.

It was also shown that, depending on the NaBH^ reduction of BR or its modified derivatives in the light or in the dark, the retinyl moiety is attached to e-amino groups of different lysine residues. Such a phenomenon is not an artifact of the reduction conditions, but is a conseguence of the photochemically aldimine migration

induced

(Fig. 1, 2).

Our experimental approach relies upon analysis of the products formed on limited proteolysis of native

(1) or reduced BR cata-

lyzed by chymotrypsin or papain. By polyacrylamide gel electrophoresis two fragments are obtained in both cases: 1-71 and 72-247

(248)-chymotryptic

papainic ones according to

fragments, or 3-65 and

(numbering according to

73-230(231)-

(2), in parenthesis -

(3)); these fragments are termed as a small and a

large one, respectively. Intense fluorescence of the retinyl residue enabled its facile detection in electrophoregrams in retinyl-bacterioopsin

and in the above-mentioned

After NaBH^ reduction of BR in the light

both

fragments.

(in all experiments

a 250 W halogen lamp was used along with 3 cm layer of 10% C u S 0 4 and yellow-orange glass filter) at pH 9.5 and 20°C retinyl moiety was found in the small and large

(4),

fragments.

Noteworthy, the proton transport across the purple membrane is

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter & Co., Berlin • N e w York

436

direction of

the

SDS-electrophoresis

+

Fig. 1. PAAG-electrophoresis of bacteriorhodopsin fragments obtained by reduction at various conditions followed by chymotryptic hydrolysis. 1) Reduction in the light followed by cleavage; 2) cleavage followed by reduction in the light; 3) cleavage followed by reduction in the dark. correlated with sequential protonation and deprotonation of the aldimine bond between retinal aldehyde and Lys 41 (see review (5)). The data obtained indicate that the e-amino group of this residue is not the only site for covalent attachment of retinal to the BR polypeptide chain and that aldimine bond may be formed by a lysine residue in small fragment (apparently Lys 41), or by large fragment residue (s) (designated as Lys x) in position 130, 159, 171(172), or 215(216). After cleavage with chymotrypsin or papain, BR preparations

437 1

< v o c 0 u) o

D

01 c £ a >s C

ffl o in c

£c

_

d i r e c t i o n of t h e

SDS-electrophoresis

+

Fig. 2. PAAG-electrophoresis of bacteriorhodopsin fragments obtained by reduction at various conditions followed by chy.motryptic hydrolysis. 1) Reduction in the presence of gramicidiri S; 2) reduction in the light at 0° and pH 10; 3) reduction in the light at 0° and pH 10. essentially preserve the spectral and functional properties of the native molecule, however acquire the NaBH^ reducibility. We capitalized on this fact to obtain information about the aldimine localization in dark conditions. In dark preparations the retinyl moiety is associated exclusively with a small fragment. After reduction of native or proteolyzed BR in the light, fluorescence was detected in both fragments. These data testify the N N retinal migration in the course of photochemical transformations of bacteriorhodopsin.

438 After dark reduction of proteolyzed BR, preliminary adapted to light or dark, or after dark reduction of preparations regenerated from proteolyzed BR with 13-cis- or all-trans retinal, all the fluorescence was found only in the small fragment. This fact means that in the dark, whatever is the double bond configuration, retinal is attached to Lys 41, and aldimine formation with a new lysine residue (Lys x) in the light is not caused by retinal isomerization during the photocycle. The BR reduction in the light was also carried out under the conditions wherein an intermediate of the photocycle (so-called M412 form or its congeners) having deprotonated aldimine bond is predominant. Stabilization of such a form is achieved by adding gramicidin S (6) OJ; by lowering the temperature (5, 7). In both cases reduction proceeds at a considerably higher rate and results in exclusive localization of retinyl on Lys x. It might be possible that under these conditions both Lys 41 and Lys x aldimines are present, but only the latter is reducible by NaBH^. However, since Lys 41-aldimine in proteolyzed BR is accessible for NaBH^ in the dark as well as in the light, localization of retinyl moiety only in large fragment when reduction is carried out in the light under the conditions favoring M412 stabilization, is indicative of the absence of Lys 41-aldimine and of complete migration of aldimine onto Lys x. Thus, a correlation exists between the aldimine bond deprotonation and its migration. The migration may proceed via aldimine hydrolysis and subsequent reaction of released retinal with another lysine amino group (earlier a photoinduced hydrolysis of aldimine bond was discovered and a hypothesis about possible migration was discussed (8)),or via a direct attack of aldimine by a spatially proximal amino group leading to tetrahedral intermediate. We found that trifluoroacetylation or succinylation of the exposed amino groups in BR fails to prevent the aldimine migration, thus allowing a conclusion to be made that Lys x amino group, inaccessible for modification, is a suitable acceptor for migrating retinal, probably being in a hydrophobic environment

439 and having a low pK. Collectively, the data on reduction of BR and its modified derivatives clearly show that migration is not a random formation of aldimines with any of accessible amino groups induced by the pH of the medium or by presence of NaBH^. The retinyl residue occupies a chromophoric site in BR when it is bound either to Lys 41 or Lys x, since none of the reduced preparations regenerates with added all-trans retinal. However, marked differences exist in microenvironment of retinyl residues attached to different lysines: a characteristic fine structure and bathochromic shift of the absorption band are seen only for Lys x -retinyl. A preference for aldimine localization on Lys 41 in the dark and a capability of photoinduced migration onto Lys x are characteristic of native BR and may be lost under certain conditions affecting the relative stability of corresponding aldimines. For example, both,after light and dark reduction retinyl is bound only to Lys 41, when analogs modified at retinal C(4) position are reduced, and only to Lys x when proteolyzed BR is reduced at acidic pH with NaBH^CN. Aldimine bond migration reported in this communication provides evidence that at least two amino groups, separated in the amino acid sequence and belonging to two different a-helical segments are involved in the BR photocycle. (Localization of these lysines is under way). This finding should be taken into consideration when interpreting the data of physico-chemical studies of BR or mechanisms of its functioning, wherein retinal aldimine is given a key role. A possibility of protonating and deprotonating different aldimines, formed by amino groups of different residues and having different microenvironment, seems very advantageous for realization of a proton transfer across the purple membrane. A photoinduced migration of aldimine bond may be a component part of the mechanism of BR functioning as a proton pump.

440 References 1. 2. 3. 4. 5.

Abdulaev, N.G., Feigina, M.Yu., Kiselev, A.V., Ovchinnikov, Yu.A., Drachev, L.A., Kaulen, A.D., Khitrina, L.V., Skulachev, V.P.: FEBS Letters 90, 190-194 (1978). Ovchinnikov, Yu.A., Abdulaev, N.G., Feigina, M.Yu., Kiselev, A.V., Lobanov, N.A.: FEBS Letters 100, 219-224 (1979) . Khorana, H.G., Gerber, G.E., Herlihy, W.G., Gray, C.P., Anderegg, R.J., Nihei, K., Biemann, K.: Proc. Nat. Acad. Sei. USA 76, 5046-5050 (1980). Schreckenbach, T., Walckhoff, B., Oesterhelt, D.: Eur. J. Biochem. 76, 499-511 (1977). Stoeckenius, W., Lozier, R.H., Bogomolni, R.A.:Biöchim . et Biophys.Acta 505, 215-278 (1979).

6.

Melnik, E.I., Chizhov, I.V., Skopinskaya, S.N., Snezhkova, L.G., Miroshnikov, A.I.: Abstracts of the I SovietSwiss Symposium "Biological membranes: structure and function" Tbilisi, p. 75 (1979) .

7.

Peters, I., Peters, R., Stoeckenius, W.: FEBS Letters 61, 128-134 (1976).

8.

Shkrob, A.M., Rodionov, A.V., Ovchinnikov, Yu.A.: Bioorgan. Khim. (Russian) 4, 354-359 (1978).

CHEMICAL MODIFICATION T o p o g r a p h y of

Horst-Dieter

OF PURPLE

MEMBRANE

Bacteriorhodopsin

Lemke, Jürgen B e r g m e y e r

Max-Planck-Institut D-8033 Martinsried,

and Dieter

Oesterhelt

für B i o c h e m i e Germany

Introduction The primary membrane

s t r u c t u r e of b a c t e r i o r h o d o p s i n

(PM) is known

The d i s p o s i t i o n was m a i n l y

(bR) in the

purple

(1,2).

of the p o l y p e p t i d e

chain first p u b l i s h e d

based on s u r f a c e c l e a v a g e sites by p r o t e a s e s

in (1) (Fig.1).

F a v o r e d by the c r y s t a l l i n e array of bR in the m e m b r a n e an ron d e n s i t y map of 7 A r e s o l u t i o n was o b t a i n e d s e q u e n c e of the p o l y p e p t i d e

chain fitted

the nonhelical density Chemical

is one a p p r o p r i a t e

p r o p o s e d a r r a n g e m e n t of the p o l y p e p t i d e by retinal

and total

insight

scattering

a p p r o a c h to test

and the amino acids near by the model

structure,

its binding

The t r i m e r i c s t r u c t u r e

in Fig.

in general

was already

(5). H o w e v e r , the sites of

were not d e t e r m i n e d ,

a l t h o u g h the knowledge of

ural

information.

chemical

represent extremely

In this article we will

modification

site, as well features

proven by

cross-

modification neighbouring valuable

summarize

of bR and the m e t h o d s

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by Walter de Gruyter &. Co., Berlin • New York

it

formed

1 and 2.

linking e x p e r i m e n t s

ami no acid residues w o u l d

the

chain. A d d i t i o n a l l y

into the c h r o m o p h o r e

as into the p r o t e i n ' s f u n c t i o n as a proton pump. Both are not e x p l a i n e d

of

(4).

modification

may give more

The

(A-G in

based on the c r i t e r i a of c o n n e c t i v i t y

links, charge s e p a r a t i o n

per helix

density

elect-

the

into this map.

a r r a n g e m e n t s of the seven regions of helical Fig.2) w e r e e v a l u a t e d

(3) and

struct-

results

used for the

of

iden-

442

Fig.

1: P r i m a r y s t r u c t u r e of b a c t e r i o r h o d o p s i n and i t s arrangement i n the membrane. P r o t e o l y t i c c l e a v a g e s i t e s of the n a t i v e p u r p l e membrane w i t h d i f f e r e n t p r o t e a s e s are marked (Ch: C h y m o t r y p s i n , P: P a p a i n , T: T r y p s i n ) ( 1 ) .

f i c a t i o n of modified

residues

i n the

sequence.

Results As a f i r s t tyrosine

reaction

residues

f o r chemical

modification

the n i t r a t i o n

by t e t r a n i t r o m e t h a n e was u s e d . A f t e r

of

subti-

443

Fig.

lisin

2: S u g g e s t e d a r r a n g e m e n t ( a c c o r d i n g to t h e m o d e l in (5)) of t h e s e v e n h e l i c e s in t h e p r o t e i n ' s t r i m e r i c unit.

digestion

complex

mixture

of t h e of

high-performance (absorbing sine

and

Tyr(N02) 3e).

nm)

nitrated

(Fig.3c),

Gly-Tyr

according

to

small

liquid

at 360

the

protein's

sequence

131

(0.6 m o l )

and

(Fig.

3f)

and

(6).

For

of

133

of

was

with of

the

of t h e the

derived,fragments

(Fig.3b),

Gly-

are 64

(1

acid

extent

integration found

hydrolysate

positions,

obtained

in

mol),

3-Nitrotyrosine

protein's

and

unique

The molar by

(Fig.

130-131)

132-133)

obtained

nitrated

3-Ni t r o t y r o -

(positions

3 moles

3f a

peptides

with

the tyrosines

was

a

Leu-Tyr(N02)

as n i t r a t e d .

tyrosine

analysis

and

(positions

(0.4 m o l )

identification

Leu-Tyr(N0?)

Ser-Tyr(N02) Val-Tyr

In F i g .

nitrated

is c o m p a r e d

identifying

each

in s u s p e n s i o n

obtained. of

(Fig.3d)

63-64),

Ser-Tyr

comparison acid

was

a digest

dipeptides

(positions (2) a l s o

of m o d i f i c a t i o n

peptides

protein

chromatogram

Val-Tyr(N02)

the

in t h e a m i n o

delipidated

from

which

by C N B r - c l e a v a g e

of

444

F i g . 3: C o m p a r i s o n of h i g h p e r f o r mance liquid chromatograms of 3 - N i t r o t y r o s i n e , nitrated d i p e p t i d e s and the s u b t i l i s i n d i g e s t of n i t r a t e d p u r p l e m e m b r a n e (6). The following samples were i n j e c t e d s e p a r a t e l y i n t o an isocratic high p e r f o r m a n c e liquid chromatography system: a) 3 - N i t r o t y r o s i n e b) S e r - T y r ( N 0 ? ) c) G l y - T y r ( N O p ) d) V a l - T y r ( N O , ) e) L e u - T y r ( N O ^ ) f) s u b t i 1 i s i n - d i g e s t of nitrated purple membrane. P e a k 6 c o u l d be r e l a t e d to a s e q u e n c e a r o u n d t y r o s i n e 26 by a m i n o a c i d c o m p o s i t i o n .

the delipidated tion

21-32

Gly-Tyr64)

protein

including a n d VII

were

analysed.

Leu-Tyr26),

(positions

V

Only

f r a g m e n t s II

(positions

118-145

61-68

including

absorption

a t 360 nm

tion

including

Leu-Tyr.47-I1e-Leu-Tyr,Rn).

146-163,

not

including

Va 1 - T y r 1 3 1 - S e r -

T y r -j 3 3 ) s h o w

but

(posi-

fragment

VIII

(posi-

445

Fig.

After

4:

reaction

soluble nm

reagent

except

longer less

S p e c t r a l c h a n g e s of p u r p l e m e m b r a n e upon nitration a) p u r p l e m e m b r a n e b) n i t r a t e d m e m b r a n e c) n i t r a t e d a n d s u b s e q u e n t l y r e d u c e d m e m b r a n e .

those

of

the n i t r a t e d

sodium

dithionite

derived

indicating

from

a reduction

3-Aminotyrosine.

membrane

As

of

accessible

to t h e

reducing

agent.

conditions

reacts

to a b o u t

20

tion

subsequent

and

spectral

changes

reduction

ionic,

absorbing

water at

detectable

3-Nitrotyrosine

to l i e o n

the

26 w e r e

a consequence surface

the

peptides

tyrosine

be a s s u m e d

In F i g . 4

the

no

with

to t h e

Tyr64,131

of t h e m e m b r a n e

and

colour-

133

therefore

3-Nitrotyrosine2g

360

any

under

can being these

%. of t h e are

purple

shown.

membrane

Nitration

upon

shifts

nitrathe

446 absorption

maximum

mum

nm.

at

360

at 3 6 0 a form does 26

absorbing show

in the

Reaction reagent mation sulting

HPLC

Reaction

of

exclusive all

64 a n d the

and

The

at 532

acid

residues

the

CNBr-cleavage Lys

phase

30

V as

and

and

of

the

subsequent

re-

ion

26

(0.25

tyrosine

64

(0.75

vesicles

of

tyrosine

64. This

oriented

identically

30 are

infor-

membrane.

tyrosine

are

26/lysine

further

of t h e

mo-

sites.

membrane-containing

modification

tyrosine

separation

II a n d

reactive

of

produce

(0.5 m o l e s ) ,

in C N B r - f r a g m e n t

trans-

(6,8).

surface

HPLC

with

strongly h y d r o p h i 1 i c

should

at t h e

remains

3-Nitrotyrosine,,g

proposed

with

nm

pK of t h i s

because

maxi-

absorption

chromophore

was

reversed

bR m o l e c u l e s

tyrosine

specific

mation

about

membrane. under

modification the

protein. the

of

located

relative therefore

lead

clearly and

to

the

demonstrates that

on o p p o s i t e

with

with

afterwards

papain

activity weight

should

upon

by t h e

S-diazotized were

be f o u n d

tyrosine

sites

of

on S D S

gels

if t y r o s i n e

on t h e

same

side

s i des.

carry

the

label

64 a n d

prepared.

amino

the

As

acids

the off

different-

carboxyterminus

64 a n d

lower the

Vesic-

(DSA) and treated

a prediction

in t h e

are

in

cleaves

experiment:

acid

of t h e m e m b r a n e if t h e y

chain

papain

infor-

electrophoresis.

following

exclusively

further

chain migrates

gel

sulfanilic

band should

with

terminal

SDS

located

chain

to o b t a i n

polypeptide

of t y r o s i n e

be d e t e r m i n e d

les c o u p l e d

the

polypeptide

chain

orientation 35

of

used

the c a r b o x y

The shortened

can

be

purple membranes

conditions

uncleaved

The

can

arrangement

Treatment

certain

1y f r o m

are

is o b s e r v e d .

the

structure

in C N B r

purple

of

a new

membrane.

This

the

located

absorption

participation

revealed

located

influences

nm,

acid by

only

creates

a t 360

at pH 8 . 8 ,

fragments

both

les)

nm

9. T h e r e f o r e

sulfanilic

amino

nm a n d

equilibrium

purple membrane

diazotized

exchange

510

pH

532

chromophore

absorption

the

Modification

that

at

chromophore of

about

moles)

the

to

reduction

A pH d e p e n d e n t

is at a b o u t not

5 6 8 nm

Dithionite

nm, w h e r e a s

unchanged. ition

from

radio-

molecular

carboxyterminus

but

located

the on

uncleaved opposite

447

1 2

3

4

Fig.5: SDS gel electrophoresis after modification of PM-vesicles with 3 %-DSA at pH 10 and subsequent treatment with papain (autoradiogram) 1) 3 5 s - m e t h i o n i n e PM.,,-

2) PM modified with S-DSA 3 5 3) PM-vesicles modified with S-DSA 4) 35S-methionine PM without papain treatment Samples 1-3 were treated with papain for 6 hours. 3S Fig.5

shows

the

result

methionine

PM s e r v e s

tide

(trace

chain

Beside

the main

lower m o l e c u l a r action of

are

3

tact

^S-DSA

containing This

the

proves

on o p p o s i t e

the

A same

to

located

the

bands

be on t h e

cytoplasmic

Unmodified

the

cleaved

(bR m i n u s

as a r e s u l t

chain

(trace

of f u r t h e r papain

2).

In c o n t r a s t ,

are

found

of

treated

tyrosine

64 a n d

the

the

the

side

in-

(trace

3).

carboxyterminus

carboxyterminus

s i d e of t h e m e m b r a n e

extracellular of

Since

sample

only

bacteriorhodopsin

papain

cytoplasmic side

if the

treated

and

of t h e m e m b r a n e .

1).

papain

and

chains

polypep-

carboxyterminus )

pattern

(trace

S-

uncleaved

S-DSA modified

are m o d i f i e d

on t h e

for

papain

product

location

sides

64

on t h e

w e i gohct

modified

shown is

4) a n d

vesicles

experiment.

as a r e f e r e n c e

reaction

seen.

PM s h o w s

of t h a t

(7),

and t y r o s i n e

was

tyrosine

26/lysine

PM.

Discussion The bR

results located

of c h e m i c a l at the

modification

surface

of a m i n o

of t h e m e m b r a n e

acid

support

residues fully

the

in

30

448 arrangement rations

gents

64,

results

reaction

reaction has

Another

light-dark could

Proteases

like

cleave

loops

reactions Tyrosine close

to

the

function

intermediate

the

nitrated to y i e l d

based

to

on

30,

rea-

the

these

exception

revealed

from the

of

by a

(6). T h i s of t h e

the

reliable

non-stoimembranes

access

to

some

heterogeneous

by e x t e n s i v e problem

in

reactivity.

sonication

membrane modification

conformational

cis-trans

reasonable

on t h e 30

states

In t h e

of

case

isomerisation

with

basis

only

It c o u l d

well

of

the

reacts

retinaloximes

for

of

reti-

the

with

soluble

26

cycle.

hydroxy 1 amine

to

be

borohydride

or

change

has

in t h e d a r k

apomembrane.

small

models.

is a s s u m e d

tyrosine

bond

sites

these

a structural

photochemical

with and

within

proposed

which

reacts

be t h a t

active

react with water

induces

for the C=N d o u b l e of

surface

of t h e

chromophore,

light which

buried

explanation

(partially)

2"6, h o w e v e r ,

membrane

to

to t h e m e m b r a n e

stages

hydrophilic

be c l o s e

non-stoichiometric

(i.e.

lysine phase.

differences.

No

lysine

in t h e

the dark

due

different

retinyliden

chromophore.

tective

close

tyrosine

hydroxy 1 amine the

such

be f o u n d

26 a n d

reagents,

reagent

of d i f f e r e n t

with

in F i g . 1 .

can

the

papain o r c h y m o t r y p s i n

very

shown

as

as a g e n e r a l

adaptation

provoke

(with the

in p a r t o v e r c o m e

be t h e e x i s t e n c e

to

However,

of m o d i f i c a t i o n

for

get

alte-

arise.

complication

reason

protein

to

little

the aqueous

be d u e to a s t a c k i n g

be c o n s i d e r e d

the membrane nal)

be

necessary with

is c o m p l e t e

excluding

This

can only to

chemistry. might

thus

may

Only

itself may

reactions

64)

in F i g . 1 .

accessible

questions

the e x t e n t

sites.

are

of t h e m e m b r a n e .

at t y r o s i n e

in s u s p e n s i o n reactive

chain

in c o n t a c t

chromophore

general

of

shown

is p a r t i a l l y

surface

several

chiometric

can

26

133

of t h e m o d i f i c a t i o n

nitration

bR

and

retinyliden

quantitation

and

polypeptide

p a t h of t h e

131

tyrosine

the

cytoplasmic None

the

of t h e

tyrosine Because

of

a

but In

already

of

pronot

at

fact, in

the

449 References 1.

Ovchinnikov, Yu.A., Abdulaev, Kiselev, A.V., Lobanov, N.A.: (1979).

2.

Khorana, H.G., Gerber, G.E., Herlihy, W.C., Gray, C.P., A n d e r e g g , R . J . , N i e h e i , K. , B i e m a n n , K.: P r o c . N a t l . A c a d . Sei. USA 76, 5046-5050 (1979).

3.

Henderson,

4.

E n g e l m a n , D.M., H e n d e r s o n , R., M c L a c h l a n , A.D., W a l l a c e , B . A . : P r o c . N a t l . A c a d . S e i . USA 7^» 2 0 2 3 - 2 0 2 7 ( 1 9 8 0 ) .

5.

Dellweg,

6.

Lemke, H.-D., 604 ( 1 9 8 1 ) .

7.

Gerber, G.E., Gray, Ch.P., Wildenauer, D., Khorana, Proc. N a t l . A c a d . Sei. USA 74, 5 4 2 6 - 5 4 3 0 (1977).

8.

Fischer,

R., Unwin,

H.G.,

N.T.:

Sumper, M.: Oesterhelt,

U., O e s t e r h e l t ,

N.G., Feigina, M.Yu., FEBS Lett. 100, 219-224

Nature

FEBS D.:

D.:

252, 28-32

Lett.

Eur.

J.

Biophys.

90,

(1975).

123-125

Biochem.

(1978).

115,

J. 2 8 , 2 1 1 - 2 3 0

595H.G.: (1979).

FORMATION AND UNUSUAL PROPERTIES OF BACTERIOOPSIN - A g + - 4DiMETHYLAMINOCHALCONE TRIPLE COMPLEX

Almaz Aldashev, Alexander Rodionov, Eugenii Efremov, Alexander Shkrob Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR

Introduction Bacteriorhodopsin

(BRh) is the main component of purple mem-

branes from Halobaoterium

Halobium

(1). Removing of the pros-

thetic group (retinal aldimine) from BRh transforms the purple membrane into colourless apomembranes containing bacterioopsin (BO) (2). Recombination of BO with,both;retinal and some of its analogs results in formation of aldimine - containing chromoproteins (3). Retinal analogs not only of aldehyde competitively inhibit BO-aldehyde recombination

nature,

(4). The Ag + -

ions are also the competitive inhibitors of the recombination and destroy the noncovalent complexes formed by BO with retinal analogs (5).

Results Apomembrane-bound 4-dimethylaminochalcone (DMCh)

(CH^)2 N _ C 6 H 4~

CH=CH-CO-CgHj. has the bands in the excitation and emission spectra (Fig. 1) at 435 nm and 500 nm, respectively

(lifetime

in the excited state ca. 2.1 nsec). The binding of the probe results in a large enhance of the quantum yield of fluorescence: this effect enabled us to calculate apomanbrane -DMCh dissociation con-7 stant (K,=3-10 M, see Fig. 2). The bound probe reveals relai i 3 2 tlvely low ellipticity in CD-spectra (|9|=4,58*10 grad-sm /

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

452

Fig. 1. Excitation and emission fluorescence spectra of DMChBO complex before and after Ag + -ions were added.

Fig. 2, 3. Determination of equilibrium constant of DMCh-apomembrane binding (C B0 =10 - 7 M) and CD-spectra of DMCh in apomembrane without Ag+ and in presence of Ag+ (C b 0 =10"5 M).

453 decimole, see Fig. 3). K^ calculated from CD data appeared to be lower than the value obtained fluorometrically. Taking into account

that spectral properties of DMCh in apo-

membrane are very similar to those of the probe, included in lipid vesicles,we can explain

the differences in K^ values

obtained by both methods so, that the main contribution in fluorescence enhancement gives the lipid-bound probe, and in CD

the protein-bound DMCh. DMCh does not inhibit the forma-

tion of BRh from BO and all-trans retinal but suppresses the recombination of BO with 13-cis-retinal (1(^=2,1-10

5

M) as well

as with tetraenal p-CH 3 0-C g H 4 -(CH=CH) 4 CH0 (K i =l,4-10 -5 M, Fig. 4). These data demonstrate the differences in the binding sites of isomeric retinals on the stage of the formation of noncovalent complexes.

Fig. 4, 5. Competitive inhibition by DMCh of binding to BO (C b0 =10 _ 6 M) of 13-cis-retinal and tetraenal (left) and all-trans retinal in presence of various concentrations of Ag + -ions (right).

454

When Ag+-ions are present even in negligible concentration, DMCh competitively inhibits the recombination of BO with alltrans retinal (Fig. 5). The Ag + -ions induce also the large bathochromic shift (40 nm) of the bands in absorption, CD and fluorescence spectra (see Fig. 1, 3). The halfwidth of the emission band

of the B0-DMCh-Ag+ complex

(AA=78+2 nm) is similar to that of DMCh in organic solvents. It means that all molecules of the probe are in the homogeneous environment. In addition to the spectral shift, Ag + -ions induce also the marked rise of DMCh ellipticity (up to |e|=16,5-10 2

grad.sm /decimole, Fig. 3), which could be due to increase of asymmetry of the probe environment. Equilibrium constant for reaction B0-DMCh-Ag+ t B0-Ag + + DMCh (Kd=3,5-10~7 M) is independent of whether it was obtained from CD or from fluorescence data (Fig. 6).vThus, Ag + -ions in small concentrations

Fig. 6, 7. Determination of equilibrium constant of binding of DMCh to B0-Ag + complex (left) . Removal of DMCh from BO by large concentrations of Ag + -ions (right), C B0 =10~7 M, MES 0,02 M, pH 6,4.

455 apparently induce not only the migration of DMCh from lipid to protein, but also modify the intraprotein binding site in such a way, that it can overlap the binding site of all-trans retinal. No other investigated metal ions cause such an effect. When the Ag + concentration is further increased, DMCh,like other retinal analogs is forced out of BO-DMCh-Ag+ complex (Kd=8-10~3 M, Fig. 7).

References 1.

Oesterhelt, D., Stoeckenius, W.: Proc. Natl. Acad. Sci. U.S.A. 70, 2853 (1973).

2.

Oesterhelt, D., Schuhmann, L.: FEBS Lett. £4, 262-265 (1974) .

3.

Schreckenbach, Th., Walckhoff, B., Oesterhelt, D.: Eur. J. Biochem. 76 , 499-511 (1977) .

4.

Ovchinnikov, Yu.A., Shkrob, A.M., Rodionov, A.V., Mitzner, B.I.: FEBS Lett. 97, 15-19 (1978).

5.

Shkrob, A.M., Rodionov, A.V.: Bioorg. Chem. 5, 376-393 (1979).

PEPTIDE FROM BEEF HEART MITOCHONDRIA INDUCING ION-SELECTIVE CHANNELS ON PLANAR BILAYER MEMBRANE

L.A. Pronevich, G.P. Mironov, G.D. Mironova Institute of Biological Physics of the USSR Academy of Sciences, Pushchino

Introduction Essential steps in studying the molecular mechanism of calcium transport regulation in mitochondria are isolation, identification and reconstruction of ion-transporting systems (1-6). This article is dealing with a new efficient method of isolation of beef heart mitochondrial peptide (P) which induces the 2+

selective Ca ranes (BLM).

-transport through artificial bilayer lipid memb-

Results 1)

Isolation of peptide. The mitochondrial fraction of beef heart tissue was extracted with 96% ethanol (200 ml per 1 g of mitochondrial protein; pH 7.0; 1 h under stirring; 4°C) and centrifuged at 5.000 x g for 15 min; 4°C. The pellet was resuspended in 50% ethanol and then extracted and centrifuged under the same conditions. The supernatants of two centrifugations were combined and evaporated to dryness in vacuum at 30°C, then dissolved in bidistilled water (1 ml per' 2 g of wet tissue) and treated with 3-fold volume of chloroform: methanol (2:1, v/v) in order to remove lipids. The lipid-free extract was evaporated to dryness in vacuum at 30°C and dissolved in bidistilled water, 1.5 ml of this solution which corresponded to 100 mg of the dry pre-

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter & Co., Berlin • New York

458 paration were applied to a Sephadex G-15 column (2.8x78 cm). The profile obtained after elution with bidistilled water (60 ml/h) is presented in Fig.l. As has been shown earlier (2), fraction

as identified by its ability to induce BLM

conductance>corresponded to the membrane-active peptide.

Fig.l. Elution profile of beef heart mitochondria,defatted water extract from Sephadex G-15 column (2.8x75 cm). Elution was carried out by bidistilled water at the rate of 60 ml/h. 2) Purification of P. Since we have shown earlier the presence of sulfhydryl groups in the peptide, further purification was conducted by means of affinity chromatography with reversible coupling of thiol-containing peptides. 150 mg of fraction 3 were applied onto an activated Thiol-Sepharose 4B column (4 ml). The non-coupling components of fraction 3 were eluted with 25 ml of 0.02 M ammonium formate

buffer (pH 8.0; 5 ml/h).

The coupled P was eluted for 4 h with 20 mM mercaptoethanol in the same buffer. The eluate was evaporated until complete removal of mercaptoethanol and ammonium formate , then the dry preparation of P was brought onto Sephadex G-15 column (2.8x60 cm) and eluted with bidistilled water (60ml/h). The 140-170 ml fraction contained P. The homogeneity of P was analyzed by paper chromatography as described elsewhere (2).

459 3)

Properties of P. The molecular weight of P as determined by gel filtration on Sephadex columns was about 2 r 000. After affinity chromatography purification the specific activity of P increases about 1000 fold. The acid hydrolysis revealed 7-8 amino acids (Fig.2). P is water- and ethanol- (70%) soluble. In water solution the Ca2+-transporting activity of P decreases at room temperature in 26 h and at 100°C in 3 min. The yield of P depended on the season as in the case of glycoprotein (3) and was 2-5 vg from 100 g wet tissue.

Fig.2. Ascending paper chromatography (Filtrak FN-1) in the butanol: acetic acid:water (4:1:5) system at room temperature: I - peptide before hydrolysis in 6 N HC1; II - peptide after hydrolysis in 6 N HC1: 1 - Leu; 2 - Phe; 3 - Ala; 4 - Pro; 5 - Glu,Gly; 6 - His,Lys; 7 - Cys 2+

4)

2+

P-induced Ca -selective conductance of BLM. Ca -transporting activity of P was tested using BLM (beef brain lipids in n-decane, 20 mg/ml) which were formed on a hole (1 mm diameter) in a teflon wall separating two chambers each containing 2.5 ml of magnetically stirred buffer solution (20 ml Tris-HCl, pH 7.5; 5-20 mM CaCl 2 ). Adding P ( final concentration!.0-5-0,ug/ml) to one or two bathing solutions of BLM one could observe in 1-5 min keeping membrane voltage constant, step-wise increases of current which were interpreted as due to formation of channels. The smallest

460 amplitudes of these steps corresponded to an increase in BLM conductance of about 20 pS (Fig.3).

C^O'ug/ml). Buffer solution contains 20 mM Tris-HCl and 20 mM CaCl2 . Besides, large jumps of conductance up to 0.5-1.0 nS were registered which persisted for tens of minutes and resembled the increase of integral BLM conductance.The higher the concentration of P, the more probable was the opening of the channels and the longer the channels remained open. Increasing the concentration of C a C ^ makes the conductance of the channels rise proportionally with the increase of the concentration of this salt. The generation of the transmembrane gradients of C a C ^ gives rise to the potential difference on the membrane (21-25 mV per 10-fold gradient at pH 6.0-8.0) that corresponds to Ca2+-selectivity of the channel. The selectivity for Ca^ + is also observed in case of large channels, which probably indicates their sieve structure, determined, in particular, by incorporation of whole P-aggregates. 2+

5)

Inhibition of Ca

-transporting properties of P by specific

agents: a) sulfhydryl groups intrinsic in P proved to be

461 essential for its ability to induce the Ca^ + transport through BLM. Addition of fluorescein mercury acetate (FMA) binding sulfhydryl groups into the BLM bathing solution results in suppressing

the ion-transporting properties of

P; b) the specific inhibitor of Ca 2 + transport in mitochondria ruthenium red (Rr) induces complete inhibition of the Ca2+-conductance of BLM in the presence of P. When one or several Ca2+ -selective channels are open, the addition of Rr (5-10~6M) results in a 100% inhibition of the effect. The

inhibition

may proceed

either jump-wise

(abrupt

closing of all or a few channels) or gradually. The inhibition rate depends on concentration of Rr. The results obtained enable the assumption that P isolated from beef heart mitochondria is involved in transport of Ca 2 + through the mitochondrial membrane. The proposal is also made that P in mitochondria functions in a complex with the glycoprotein.

References 1. 2.

3. 4.

Carafoli, E., Sottocasa G.: Dyn. Energ. Transducing membranes, Amsterdam, 455-465 (1974). Mironova, G.D., Pronevich, L.A., Fedotcheva, N.I., Sirota, T.V., Trofimenko, N.V., Mironov, G.P.: Mitochondrial processes in time organization, Pushchino, 126-140 (1978). Mironova, G.D., Sirota, T.V., Pronevich, L.A., Trofimenko, N.V., Mironov, G.P., Kondrashova, M.N.: Biofizika, 2_5, 276-280 (1980). Azarashvili, T.S., Lukjanenko, A.I., Evtodienko, Yu.V.: Biochimia, 47, 1139-1142 (1978).

5.

Lunevsky, v., Zherelova, 0., Alexandrov, A., Vinokurov, M., Berestovsky, G.: Biofizika, 25, 685-691 (1980).

6.

Sottocasa, G., Sandri, G., Panfili, E., de Bernard B., Gazzotti, P., Vasington, F., Carafoli, E.: Biochem. Biophys. Res. Commun. 47, 808-813 (1972).

FUNCTIONAL ROLE OF THE PROTEIN COMPONENT OF THE Ca 2 + TRANSPORTING GLYCOPROTEIN FROM BEEF HEART HOMOGENATE AND MITOCHONDRIA

T.V.Sirota, L.A.Pronevich, G.D.Mironova Institute of Biological Physics of the Academy of Sciences of the USSR, Pushchino, Moscow Region, USSR

Introduction 2+

Recent investigations have shown that the Ca -transporting system of mitochondria contains substances of glycoprotein origin (1,2). A confirmation of this statement is provided by our data that mitochondria contain a glycoprotein (GP) with a molecular mass 40 kD, which, when reconstructed on a bilayer lipid membrane (BLM), activates a selective transport of calcium ions (3). There is some evidence in literature showing 2+

that Ca

-transporting activity may be due to phospholipids

(4). The aim of the present work was to elucidate whether the phospholipids enter into composition of GP and what part of the GP molecule is responsible for its ionoforic properties. Results The attempt to identify the lipid component in the fraction of GP after electrophoresis in polyacrylamide gel (PAGE) by means of sudan black has not given positive results. At the same time our experiments have shown that the amount of inorganic phosphorus (P^) in GP is 13.6 yg/mg of the protein whereas the preparations of GP isolated by the other authors (1,4) contain up to 300 yg of P^ per 1 mg of protein. The disparity in the results may be probably accounted for by the fact that in our experiments the partially purified preparation was treated

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter &. Co., Berlin • N e w York

464

with chloroform-methanol (2:1). After additional special treatment of GP we managed to isolate some amount of lipids using the method of thin-layer chromatography (TLC). At the first stage of extraction conducted by the method of Folch (8) negligible amount of neutral lipids was found: fatty acids, mono-, di-, triglycerides, cholesterol as well as the phospholipids phosphatidylserine and lysophosphatides. Following the second stage of GP treatment conducted by the method of Blondin (9) much less amount of the same neutral lipids and phospholipids was isolated. GP obtained after the two-stage treatment with organic solvents does not contain any lipids. Simultaneously with determination of the lipid composition, at 2+ each stage of lipid extraction the Ca -transporting activity of GP and the effect of ruthenium red (Rr) was studied by reconstruction on BLM. The results are presented in Table 1. As 2 + seen from the Table, after removal of lipids from GP its Ca -transporting activity does not change, the inhibiting effect of Rr, the specific inhibitor of calcium transport in mitochondria, being retained. The data obtained by PAGE indicated that after all stages of treatment GP contains, both, the protein and carbohydrate components. As shown by PAGE GP does not change prior and after treatment with organic solvents. This indicates that removal of lipids does not result in denaturation of the protein and that the contribution of the lipid component to the molecular mass of GP is insignificant. The data that treating with 2+the mixture butanol:acetic a,cid:water does not affect the Ca transporting activity have been obtained earlier (3) when using the method of ascending paper chromatography in the same solvent system. The obtained results indicate that lipids are not essential for functioning of GP. It is likely that lipids, namely the phospholipids prevent a fast incorporation of GP into a model membrane, since GP from beef heart mitochondria containing minor amount of lipids starts to affect BLM conductance in 2-5 rain (3), whereas GP from rat liver mitochondria containing a great amount of lipids in 40-50 min (4).

465

Table I. Ca extraction.

2+

-transporting activity of GP after lipid

Preparation

GP,pg/ml

BLM conductance -1

(Ohm Control (without GP) GP initial

20

GP after Folch procedure

20

GP after Folch and Blonding procedures

20

Rr inhibition,

-2

-cm

)

per cent

(8.2+1.0)-10 (7.3+2.0)-10~7

100

(6.2+2.5)-10~7

100

(7.1+3.1)-lo"7

100

Media - tris-HCl pH 7,5 - 10 mM, CaCl2 - 10 mM, Rr - 5-10~6M 2+

Table 2. Proteinase treatment effect on Ca activity of GP . Preparation

GP yg/ml

-transporting BLM conductance (Ohm- 1 'cm - 2)

Control (without GP) GP initial GP after proteinase

20

(6,8+1.4)•10-9 (5.5+2.0)•10~7

treatment

20

(8.8+3.2)•10~9

without proteinase

20

(5.4+4.2)•10~7

Proteinase after incuba,tion

-

(6.5+1.5)-10-9

GP after incubation

Medici as in Ta,ble 1.

466

Further evidence for the essential role of the protein component in the function of GP are the experiments with proteinase (Table 2) . As seen f;rom the Table, proteinase-treated GP loos2+ es completely its Ca -transporting activity. Thus, it follows from the results presented in the paper that the main functional role in GP is played by its protein component, When reconstructed on BLM by means of lipid-free GP the 2+

Ca -transporting system is inhibited by Rr. This enables the assumption that it is GP (its protein moiety) that is the main component in the system of electrogenic influx of calcium into mitochondria. The phospholipid isolated from rat liver GP whose Ca 2+ -transporting activity is not inhibited by Rr is pro2+

bably involved in the system of Ca

efflux from the mitochond-

ria which exists in the mitochondrial membrane.

References

1. Carafoli,E., Sottocasa,G: Dyn.Energ.Transducing membranes, Amsterdam, 455-465 (1974). 2. Moore,C.: Biochem.Biophys.Res.Commun., 42^ 298-305 (1971). 3. Mironova,G.D., Sirota,T.V., Pronevich,L.A,, Trofimenko,N.V., Mironov,G.P., Kondrashova,M.N.: Biofizika, 25, 276-280 (1980). 4. Evtodienko,Yu.V., Medvedev,B,I. , Yaguzingsky,L.S., Azarashvili,T,S., Lukjanenko,A.I., Kuzin,A,M.: DAN SSSR, 249, 1235-1238 (1979), 5. Reynafarje,B., Lehninger,A.; J,Biol.Chem., 244, 584-593 (1969). 6. Jeng,A., Ryan,E., Shamoo,A.: Proc.Nat.Acad.Sci.USA, 75, 2125-2129 (1978). 7. Tyson,C.A., Lande,A.V., Green,D.E.: J.Biol.Chem., 25, 1326-1329 (1976). 8. Folch,J., Lees,M,, Stanley,J.: J.Biol.Chem., 226, 497-509 (1957). 9. Blondin,G.: Biochem. Biophys, Res. Commun., ¡56, 97-105 (1974),

OPTICAL SPECTROSCOPY STUDY OF SUBSTRATE BINDING BY LEUCINE SPECIFIC AND LEUCINE - ISOLEUCINE - VALINE BINDING PROTEINS FROM

E. eoli

Igor Nabiev, Sergey Trakhanov, Alexandr Surin, Tatyana Vorotyntseva, Evgenii Efremov, Vladimir Pletnev Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR

Introduction Leucine - isoleucine - valine-binding

(LIV) and leucine speci-

fic (LS) proteins are the components of high affinity transport systems for branched chain amino acids in E. aoli K12. These proteins are located in the periplasmatic space being released on cold osmotic shock procedure (1). The polypeptide chain of the LIV protein consists of 344 amino acid residues including 3 Trp, 13 Tyr, 9 Phe and one S-S bridge (2). The homogeneous LIV protein contains tightly bound substrates as L-leucine, L-isoleucine and in less amount L-valine which might be released by reversible denaturation treatment in urea solution with subsequent urea removal. In contrast to LIV, the LS protein can be obtained in crystalline form

free of L-Leu

without the treatment mentioned above. The primary structure of the LS protein which is not completely determined yet (the most probable number of amino acid residues is 346, including 4 Trp, 13 Tyr

and 9 Phe residues) revealed a high degree of

homology with the primary structure of the LIV protein (3).

Results Recently it has been shown that binding L-Leu, L-Ile and L-Val

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter & Co., Berlin • N e w York

468 causes changes in optical spectra of the LXV protein (4). The positions of the maxima and fluorescence band shapes of LIV and LS proteins point to differences in polarity of Trp environment with at least one Trp buried in the protein interiorThe changes of the fluorescence spectra of LIV and LS proteins upon substrate addition (Fig. 1) reflect different environments of the Trp residues in the complexed with respect to the free forms of the proteins. In case of LIV the shape of the differential spectra depends on the type of substrate (4). The ions I and Cs + quench significantly the fluorescence of the Trp residue (-s) in the Ls protein but do not influence the fluorescence of the protein. In the L-Leu complex of LS the fluorescence quenching is less pronounced. It was suggest-

Fig. 1. Fluorescence spectra (F) LIV (1) and LS (2) and relative changes in fluorescence (AF) LIV (3) and LS (4) upon L-Leu complex formation. ^ e x c = 2 9 5 nm, protein concentration 5 yM, L-Leu concentration 10 yM.

469 ed that a Trp residue (-s) on the surface of the LS globule becomes less accessible to the solvent environment as a result of complex formation. Differential UV spectra obtained upon substrate binding by LIV and LS proteins reveal changes in the aromatic residue environment due to complex formation. The common feature of differential spectra of LIV and LS proteins is the presence of bands corresponding to Tyr and Phe chromophores which might be interpreted as a transfer of the side chains of these residues into a more hydrophobic environment. Contrary to LIV in LS protein the bands at 290 and 300ratiassociated with Trp residue (-s) are clearly revealed. These bands are not observed in the LIV protein spectra. Probably the

Fig. 2. Differential UV spectra of LS (1) and LIV (2) after L-Leu addition. Protein and substrate concentrations are 5 yM and 10 pM.

470 Table 1. The amount of Trp and Tyr residues accessible for solvent in LIV and LS proteins and in the L-Leu - protein complexes.

Protein/Perturbant LIV LIV+L-Leu LS Ls+L-Leu

Ethylene glycol 20% v/v

Sucrose 20% w/v

Tyr

Trp

Tyr

Trp

7 5-6 9

2 2

7 5-6

2 2

5-6

3 1-2

charge alteration in the vicinity of the chromophore causes the appearence of a minimum at 300 nm. The solvent perturbation differential UV spectroscopy has been applied for quantitative estimation of the effect of substrate binding on the solvent accessibility of the aromatic amino acid residues (Table 1). In comparison with LIV the number of accessible Trp residues of LS protein is diminished of at least one residue. In the CD and Raman spectra practically no changes are observed upon substrate binding. Complex formation does not obviously affect the secondary structure of the polypeptide chain in the LIV and LS proteins. Comparison of differential UV, fluorescence, CD and Raman spectra with the data of primary structure of these proteins enables us to suggest that Trp-18 (for the LS protein) and Tyr—18 (for the LS protein) participate in the formation of a substrate binding center. A model of the binding was also proposed (4). The confirmation about participation of Tyr, Trp, Phe residues in complex formation has been obtained from the comparison of surface - enhanced Raman spectra of the free and complexed LS protein adsorbed on a silver electrode. The changes of the

471

Q.

Fig. 3. Surface - enhanced Raman spectrum of LS protein (B) and LS+L-Leu (A) adsorbed on a silver electrode. Protein concentration 0.5 mg/ml, X = 632.8 rati.

surface - enhanced Raman spectrum on substrate binding suggest that the LS protein maintains its ability for substrate binding even after adsorption on the electrode surface (Fig. 3).

References 1. 2.

Oxender, D.L.: In Biomembranes chap. 2 (Manson, L.A., ed.), New York, Plenum Press 25-79, 1974 Ovchinnikov, Yu.A., Aldanova, N.A., Grinkevich, V.A., Arzamazova, N.M., Moroz, I.N.: FEBS Letters 7^, 313 (1977).

3.

Soldatova, L.N., Shakhparonov, M.I., Chertova, E.N., Aldanova, N.: In Abstracts of 5th Ail-Union Symposium on Chemistry and Physics of Peptides and Proteins. Baku, 124,

4.

Vorotyntseva, T.I., Surin, A.M., Trakhanov, S.D., Nabiev, I.R., Antonov, V.K.: Bioorg. Khim. 7, 45 (1981).

1 980

INVESTIGATIONS OP IMMUNOGLOBULIN STRUCTURE IN SOLUTION

Vladimir Zav'yalov,Vyacheslav Abramov,01ga Loaeva,Vladimir Tishchenko,Elena Dudich,Igor Dudich All-Union Research Institute of Applied Microbiology, 142200 Serpuckov,Moscow Region,USSR Frantisek Franek Institute of Molecular Genetics Prague 6,Czechoslovakia George Medgyesi Institute of Haematology H-1502 Budapest,Hungary

Introduction The available data on the three-dimensional immunoglobulin structure have been obtained as a result of X-ray structural analysis of some human myeloma immunoglobulins G belonging to the first subclass (IgG1 Dob,Col,Meg) and of immunoglobulin A produced by murine plasmacytoma (M 603) (1).These data give a reason to conclude only - the most general regularities of the spatial arrangement of polypeptide chains of immunoglobulins G.In particular,these data manifest the conformational mobility in the so-called "switch" and "hinge" regions. It is likely that during crystallization only one of the possible dynamic conformational states is fixed.The equilibrium in solution may be shifted towards the other conformer,and it may appear to be the decisive factor to understand the functional immunoglobulin differences and the mechanism of activation of their effector functions.Besides,it is known that even immunoglobulins G of different subclasses differ significantly in their functional properties (2).The conformatio-

Chemistry of Peptides and Proteins, Vol. 1 © 1982 by W a l t e r de Gruyter &. Co., Berlin • New York

474

nal basis of these differences is still unclear,though the main differences in the primary structure of immunoglobulin G subclasses are known to be concentrated in the "hinge region" (3).One may suppose that the structure of this region can affect the functional properties of immunoglobulin G subclasses ,both, by influencing the degree of relatively independent motions of Fab and Pc subunits in solution and by influencing the structure of Pc subunit

and,correspondingly,the

conformation of effector sites.In order to verify this supposition, we have investigated in the present paper the normal rabbit immunoglobulin G,the homogeneous rabbit antibodies to pneumococcus polysaccharides,precipitating (early) and nonprecipitating (late) pig antibodies to dinitrophenyl derivatives and homogeneous human myeloma immunoglobulins G of 4 subclasses.The investigations were carried out with the help of the complex of physical methods allowing to estimate the conformational properties of proteins in solution (differential temperature- and solvent-perturbâtion spectrophotometry, adiabatic scanning microcalorimetry,polarization of fluorescence, spin label method of EPR,method of proton relaxation of NMR,circular dichroism) and also the electronic microscopy.

Results 1) Immunoglobulins G of different species origin and of different subclasses differ significantly not only in the character of interactions between Pab and Pc subunits H pppG • ppVJ • POpA jh» f+0+^pppGp"Up*UpGpA(iUpApGpAp*U RINA - polymerase ^l »

cvm

I,« in5

Fig. 1. Crosslinking of the RNA polymerase with photoreactive RNA in transcribing complex. (1)-after irradiation. (2)-without irradiation. (3)-the complex was destroyed before the irradiation.'

active label covalently bound to RNA polymerase upon illumination at X >290 nm may be due only to the oligonucleotides synthesized in the system, rather than to initial substrates, because the y-azidoanilide of GTP is not radioactive, whereas 32 the pp pU is not photoreactive. After synthesis and irradiation the reaction mixtures were subjected to SDS electrophoresis. The slabs were stained with Coomassie and autoradiographed. After photoaffinity labeling (two triphosphates are present) the B,B' and a-subunits become radioactive (Fig. 1 left-hand side), but in the case of three substrates (right-hand side) radioactivity predominates in g,B'-subunits, which presumably reflects the shift of the 5'-end of the nascent oligonucleotide within the transcribing complex when its length grows continuously up to decanucleotide. A similar methodology was employed to establish the subunits contacting with the 3'-0H end of the growing RNA. Affinity modification was made by the oligonucleotides being synthesized

487 in situ. They contained photoreactive 5-halogenopyrimidine residues in the vicinity of 3'-OH end. In the presence of GpU (the primer), 5-IU or 5-BrU and |a-32p| GTP (substrates) and the above-mentioned X imm DNA fragment (template) only cr-subunit incorporated the radioactive label upon UV-irradiation (3) . During primary structure determination of B-subunit (see V.M. Lipkin et al., this volume) the limited tryptic hydrolysis was made. Separation and sequencing of low molecular weight peptides

together with their yields

enabled to find regions of

polypeptide chain easily accessible for the protease. Two regions in the middle part of the polypeptide chain (amino acid residues 528-548 and 678-758) and C-terminal part of the molecule (residues 1105-1342)are evidently on the surface of the B-subunit and possibly represent flexible connections between stable domains. A topographic study of the RNA polymerase a-subunits was made by modifications of RNA polymerase with iodoacetate, iodoacetamide, mercury ions and K^lFeiCNjgl. The results are summarized in the model (Fig. 2). jyftiCNy

Fig. 2. Scheme of actions of different chemical agents on sulfhydryl groups of the RNA polymerase a-subunits.

488 2*

IHg

V 6-W

>

2+

IHg 2.DTT HCHjCOOH 2.Hg UCHjCONHj 2.Hg

* ¥

t -

• »

I.KgtPefCNIg]

* *

Fig. 3. SDS electrophoresis of the modified RNA polymerase. The order of modifications indicated. Buffer: 0.05 M Naphosphate pH 7.0, 0.1% SDS.

The assignment of the alkylations to definite residues of the a-subunit polypeptide chain was made by the analysis of the a-subunit sequence (4) and electrophoretic mobilities of labeled peptides of tryptic digest on thin-layer peptide maps. 2+ Formations of intrasubunit (Hg ) and intersubunit (disulfate bridge) crosslinks were shown with the aid of SDS electrophoresis (Fig. 3).

References 1.

Ovchinnikov, Yu.A., Efimov, V.A. , Chakhmakhcheva, O.G., Skiba, N.P., Lipkin, V.M., Modyanov, N.N.: Bioorg. Khim. 5, 1410-1421 (1979) .

2.

Sverdlov, E.D., Tsarev, S.A., Kuznetsova, N.F.: FEBS Letters 112, 296-298 (1980).

3.

Sverdlov, E.D., Tsarev, S.A., Begar, V.A.: FEBS Letters 114, 111-114 (1980).

4.

Ovchinnikov, Yu.A., Lipkin, V.M., Modyanov, N.N., Chertov, 0.Yu. , Smirnov, Yu.V.: FEBS Letters 76_, 108-111 (1977).

STRUCTURE

Jürgen

OF

THE

PROTEOLIPID

Hoppe

and

Walter

SUBUNIT

OF

THE

ATP

SYNTHASE

Sebald

G e s e l l s c h a f t für B i o t e c h n o l o g i s e h e D-3300 Braunschweig, Germany

Forschung

mbH.

Introduction

Membrane show are

bound

common

of

activity

+

H -conduction energy from

the

fied of

about

protein and

ATPase

is

was

referred

The

inhibited some

rum

[11,13] .

which

to

found

as

lead

to

and

from

of

the

exists

be

subunit

antibiotics activity

numerous

been

as

inhibitor

of

to

be

synthases e.g.

resistancy

or

to

C h e m i s t r y of P e p t i d e s a n d P r o t e i n s , V o l . 1 © 1982 by W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k

acid

butanol

cow.

a

[10] the

ATP

But

from

proteolipid synthase target

from

of

se-

oligomycin mitochondria

R h o d o s p i r i 1 1 urn

subunits

p.4-20 ]. A m i n o

and

the

oligomycin.

as

identi-

protein

hydrophobic

to

appeared

ATP

a

was

inhibited

The

bound the

proteolipid

sequenced

Beechey

of

bacteria,

were

is

or w i t h

(DCCD)

a subunit

e.g.

[5-8] .

a h e x a m e r [9 1. T h i s

a6

mitochondria. covalently

for

isolated

subunits

chloroform/methanol

heart to

catalyzing

E. coli

part

the

necessary

subunits F^

They

bears

F^,

been

different

different

[1-4].

are

has

organisms

which

part

parts

part

a proteolipid.

identified

the

years

have

two

likely

with

photosynthetic

recent

three

most

proteolipid

and

PS-3

of

constituent

in b e e f

hydrophobic

ganisms

coli

Both F^

dicyclohexylcarbodiimide

was

later

membrane.

bacterium

F^,

integrated

functional

composition

which

that

membrane

the A

major

activity

j1—4].

In

the

extractable

carbodiimide

only

part

In E .

8000d is

thus

veral

properties

subunit

discovered

which

functional

and

The

various

associated

across

in P S - 3 .

from

and

thermophilic

[6-8],

isolated

a membrane

transduction.

A minimum found

synthases

structural

composed

ATPase

ATP

rub-

various

or-

substitutions

a nonfunctional

enzyme

490 have been

determined Jl4,17,18,20,21].This

fore concentrate this protein, carboxyl

on the description

especially

residue which

based

to be intimately

for the conformation

on C D - m e a s u r e m e n t s

will be

of the primary

on the identification

seems

ton c o n d u c t a n c e . A model

article will

and secondary

there-

structure

of an

involved of the

structure

of

invariant in

pro-

protein

prediction

discussed.

Results General

properties.

by extraction graphy

The p r o t e o l i p i d s

with c h l o r o f o r m / m e t h a n o l

on c a r b o x y m e t h y l - c e l l u l o s e

acid

degradation

{14-21] .

residues which

weights

determined

containing lubility

only

at the N - t e r m i n u s Long found of the

both

the

25 amino

as well

hydrophobic their

so-

clustered

of the protein

s t r e t c h e s of about

amino

molecular

explain are

of the N e u r o s p o r a

of the s e q u e n c e s

the numerous conserved be i n d i s p e n s a b l e

ance of certain

structure

(residues

crassa acid

sequence).

residues

as in the C - t e r m i n a l

of these h o m o l o g o u s amino

acid

are part

the middle

of the N - t e r m i n a l

gln^g, p r o ^

residues

localized

found

to be invariant.

basic

amino

dic amino

acid

acid

for the function

hydrophobic

subunit

hydrophobic

in the h y d r o p h i l i c (arg^)

stretch

(position

maintainhomologies

(residues arg^»

segment were The only one

In 27

asn/

also

conserved invariant

in the middle of the 65). With

re-

resi-

were found.

the single

occurs.

is observed

stretch

These

or the

are c o n s e r v e d . G l y ^ »

In this segment

residue

residue

proteins

residues.

of the protein. E x t e n s i v e

the s e q u e n c e s of the proteolipid

- 3 3 ) four glycine

minal

residues

in the middle

in the N - t e r m i n a l

dues might among

and

residues which Polar

phase

72 - 82

sequence.

A comparison vealed

is in a g r e e m e n t with

solvents.

chromato-

solid

contain

on S D S g e l s . They are extremely

the numbering

hydrophobic

subsequent

by automated

The proteins

16 - 25% polar

in organic

40 - 50 using

number

and

purified

in c h l o r o f o r m / m e t h a n o l [14-21].

All the s e q u e n c e s were determined Edman

can be generally

aci-

C-ter-

exception

491

Ni'urospora cniyüu

r"i i — lyi -Sei-'jc'i-Glu-l h -Aki-Llii-rtla-Mrt-V^i-Lju-\Jul-bL'i^Lys-lAi,N-Leuilily .Het-JClyjbtir-Ala-Ala-Ile-jGly

Aup-l li'-Asp-lhr-Ala-AlaiLys

GlylAla-Ala-Thr-Val-JUy.

Cly

t -McL-Uln-L tiu-Vul-Lcu-Ala-Alo-Lys. lyr-Ile Cly Ala-tly-I le-Ser-Thr-lle*ly.

I ' Ala-tly'Leu-Ala-Ala-Ue-'ClyJMett-Mi-t-Aiip-Ala-Glu-Alu-Ala-L ys-Mot-l li --J I I L..I ! I

HMuduspu'i i lum l ubru

1 I I t -Met -Asn-l'iu-l l'u-I le-Ala-Ala-Ala-Scr-Vul-l lelAla.Alo-Cly-Leu-Ala-ValJGly-Leu-Ala-SerI i I I I I

Spinach

r-nct-Asp-l'io-Li-u-Ilc-SiT-Alo-Ala-Scr-Val-Lcu-All Alo-«la-Lcu-A]a-Ile-iCly-LiHjÍAla-AlaI >

Mast njocladus l m m n u s u s

i f-Net-i;iu-Asn-Leu-Asn-Mi't-Asp-Leu-Lcu-Tyr-Met.»All

Acidu caldarius

I I « , i r--> Val-Met-HetJCly-LeujAJa-IA]«I I

I I

I

I I

Ala-

f-MeL-Gln-Lcu-Asp-Met-Val-Lys-Ala-1le-Tyr-Asp-Ile-Ala. 1

Ile-Ala-Val-Cly-LeuÍGiy AI«.; J

f"-Het-Ser-Leu-Gly-Val-Leu-»Ala

Ncuruspora crassa

SO r—i Tyr-Gly Ala- Gly lie.Gly Ile. :iy L eu-Va1-Phe-A1a-AlaJL euM. eu-Asn-ily- Val-Ala- Arg-Aan-Pro iAla-Leu^Arg^ I I Ala-Cly. •Ser- Cly -Ala. Gly lie. Cly Thr-Val-Phe-Cly-Ser-lLeuille-lle- Gly. Tyr-Ala- Arg-Aan-Pro !Ser-Leu4.ya* I

Saccharnmyces cerevisiae

Leu-Gly

Gly lie. Gly

Rhudospirillum rubrun

lie Gly

Gly

lie Gly Pro. Cly.

Ile-Val-Phe-Ala-Ala.

Ile-Asn- Gly. Val-Ser Arg-Aim-Pro ! .Ser-lle^Lya.

Gly. .Val. Gly Asn-lle-Trp-Ala-Asn-Leu-1le-5cr.

Val-Cly. -Arg-Aao-Pro. Ala-Ala-Lys

Gly. -Gin Gly Thr-Ala-Ala-Gly-Gln.Ala-Val-IClu.lGly lleJAla. Arg-Gln-Pro Clu-Ala-GluI I I I

Mastigocladus laminosus

I le-Gly Pro Gly lie Gly •Gin- Cly Asn-Ala-Ala-Gly-Gln-Ala-Val-Glu-jCly.

Gly

lie.Gly

Cly

I - Cly..Ala-lAla. Arg-Gln-Pro. Asp-lLeu-lleI I I

Acido co Idarius

Gly •Ser Gly Val Cly. Asp. Gly Met-Val-Met-Ser. Lyallyr-ValJClu-i- Gly Val-Ala- Arg-Gln-Pro. Clu-Ala-ArgI I I I I Leu Gly Ala- Gly 1 le. Cly

Cly. Leu-Ile-Val-Ser-lArgiThr-Ile-Clu. i- Gly. J

Arg-Gln-Pro- Glu-Leu-ArgI

492 60 I" • i r-i r * •> Gly-Gln-Leu-Ptic-Scr-Tyr-Ala-I lc-LeujGly-Phc •I'he-Val- Glu-Ala .LeutMet'Vel I i i I i i I Uovine C ln-i; ]n-( ru-l'lii'-Sur-1 y r-A]a-1 le-L eu-fi 1 y-Phc :iu-Alafj'let-GlyiLcu-PhejCys Leu-iMet-'l/allAlaI I I I < Saccharomyces ccrevisiau Asp-Thr-Vul-l'hr-Pro-Mut-Ala-llc-Lcuiciy-Phe Ihr-GlyJleu-PheACys1 1 I I Rhodospirilium rubrum 5er-lhr-Vul-i;iu-l cu-Tyr-Uly-Trp-l li^fiì 1>-IMie Ile-AlaJLeu-Phe-Ala-Leu-Val-W 1 L-J c -

Ncurospora crassa

Spinach

lily-l.yu-l li'-Anj-(;iy-Ihr-lru-Lc'u-Lru-SL»r-Leu

Mast Kjocluduii 1 aminosuu

Acido caldarius

Lcu-Thr-Ile-Tyr-Gly-Leu-Val-Val-Ala-

Gly-Lys-I le—Ari)—ti]y—Ihr—Lcu—Lcu—Leu— Ihr—I cui

Leu-Thr-Ile-Tyr-Gly-Leu-Val-Ile-Ala-

Pro-Leu-lcu-Arg-Thr-Cln-Phc-Phe-1lc-Val-Met •CI»

r-T |--1 Asp-Ala I le-jProiMet-fl leAla-Uol-Gly-Leu-Cly-

Gly-Ser-Ilc-Phe-Gly-Scr-Ala-Lcu-Leu-Gly-Ual

•Valille.Ala-Leu-Ala-Phe-GlyI I Ile-Ile-Gly-Val-Val-Phe-Ser-

Pro-Val-Leu-Cln-Thr-Thr-Mel-Phe-Ile-Gly-Val

Neurospora crassa

l .J

-J

Leu-Met-Ala-Lys Phe-Leu-Ile-Leu-

Saccharomyces cerevisiae Phe-Leu-Leu-Leu