Protein Structure and Protein Engineering

Transcription

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2 39. Colloquium der Gesellschaft for 8iologische Chemie April 1988 in Mosbach/8aden Protein Structure and Protein Engineering Edited by E.- L. Winnacker and R. Huber With 60 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo

3 Prof. Dr. ERNST-LuDWIG WINNACKER Labor fur Molekularbiologie, Genzentrum, Am Klopferspitz, 8033 Martinsried, FRG Prof. Dr. ROBERT HUBER Max-Planck-Institut fur Biochemie, Am Klopferspitz, 8033 Martinsried, FRG ISBN-13: DOl: / e-isbn-13: Library of Congress Cataloging-in-Publication Data. Gesellschaft fur Biologische Chemie. Colloquium (39th: 1988: Mosbach. Baden-Wurttemberg, Germany) Protein structure and protein engineering/edited by E.-L.Winnacker and R. Huber. p. cm. 1. Proteins-Structure-Congresses. 2. Protein engineering-congresses. I. Winnacker, Ernst L. II. Huber, R. (Robert), III. Title. QP551G '245-dc This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its version of June 24, 1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law. c Springer-Verlag Berlin Heidelberg 1988 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Printing and binding: Bruhlsche Universitiitsdruckerei, Giessen 2131/

4 Preface Protein engineering has had considerable impact on basic and applied research in biochemistry and molecular biology. It is already in use as a tool in molecular biology, but it is beginning to strongly influence the planning of experiments in biology everywhere, and, with even further-reaching consequences, the appointment politics in research institutions and industries. Protein engineering, perhaps more than any other methods of protein analysis and peptide synthesis, has shown that proteins are organic molecules governed by the universal laws of chemistry and physics. However, as was the case with other new powerful methods and techniques, protein engineering tempts to an exploration of its limitations and thus generates more questions than it answers. The 39th Mosbacher Colloquium on Protein Structure and Protein Engineering is not the first conference on this topic and it will not be the last. The important issues are obviously techniques of protein engineering, examples of application, and the basic framework of protein structure and stability within which reasonable experiments can be designed; conversely also, what we can learn about protein structure, dynamics, and folding from such experiments. Experiments in this direction aim at elucidating the folding code in the long run, but help to exploit the role of individual amino acid residues in catalysis, protein stability, and binding specificity in selected proteins now. Consequently, there are already practical applications of enzymes engineered for increased thermal stability (Alber), or of protein protease inhibitors with deliberately modified specificities (Bode). These experiments have to adhere closely to the parent molecules in structure and function to allow a rational approach on the very limited basis of our knowledge of protein structure and function, but the goal, or rather dream, is to design novel proteins with given specified functions. This goal reaches even further than the solution of the folding code and there are doubts whether it is realistic. It may be realistic for a particular class of proteins constructed from a framework and hypervariable segments which can adopt virtually any structure, the immunoglobulins. Protein engineering with immunoglobulins follows nature, which makes use of gene rearrangement to generate virtually unlimited binding specificities. The protein engineer's approach is either to use the natural genetic repertoire and the methodology of monoclonal antibodies (Lerner) or to graft artificial amino acid sequences onto immunoglobulin frameworks (Pllickthun). In view of the many facets of protein engineering and-within the given narrow time frame, the scientific program had to be quite selective and different aspects of protein structure, stability, and dynamics and different exemplary protein systems could have been chosen. The organizers nevertheless wanted to document the concepts on which experiments of protein engineering must be based and to demonstrate the kind of results expected or obtained. The experiments are rationalized in terms of protein structure so that this aspect played a central role. The forces that hold proteins in a defined three-dimensional structure

5 VI result from intramolecular interactions in the polypeptide chain and from intermolecular interactions with the solvent. The study of the interplay of solute and solvent and the underlying thermodynamic parameters is a classical field, but provides new insights when modern techniques and theoretical analyses are applied (Privalov, Dill). The protein solvent system is by far too large for a quantitative evaluation of the time-independent Schrodinger equation involving electrons and nuclei, or, derived from it, for semi-empirical and abinitio methods of quantum chemistry. Molecular dynamics, however, which treats molecular systems as ensembles of atoms with pairwise interactions according to empirical potentials, is feasible even with presentday computing facilities and holds some promise of reproducing experimental protein structures and thermodynamic properties (Levitt). A fully empirical approach to protein structures has gained considerable importance (Jones). It uses structural data from known proteins to suggest folds of segments in unknown proteins. It is based on the observation that despite the versatility of protein structures, local geometries are conserved (canonical a-helices, S-structures, turns, etc.). Proteins have stable structures essential for their functions but their dynamic properties seem to be equally important. Molecular motions are involved in molecular interactions at all structural levels of residues, segments, and domains (WUthrich, Clore, Huber), not only in proteins but surprisingly also in DNA (Sigler). The model of the DNA double helix gives an impression of stiffness, which the real molecule does not have.. The problem of the pathway from the unfolded to the folded state is experimentally extremely elusive, as the intermediates are unstable and short-lived. It is even unclear whether there exists in general a unique pathway or whether renaturation proceeds by many routes (Jaenicke). Renaturation experiments of engineered protein variants add a new dimension to these studies which may finally provide an answer. There exists an alternative to protein engineering by recombinant DNA technology: chemical synthesis. with the availability of automats and high resolution separation methods chemical synthesis has considerable advantages in the synthesis of variants by fragment condensation (Kaiser, Bayer). It has the unique possibility of introducing unnatural components and exploiting the features of such groupings (Bartlett). Automatization has also advanced DNA sequencing, the prerequisite of protein engineering. The analysis of the genomic structure of the human being is thus becoming a realistic goal (Wada). Ultimately we wish to understand protein structure and function. The latter concerns the thermodynamics and kinetics of its interaction with substrate. These reactions may be very complex and involve conformational transitions, but can be analyzed in quantitative detail. The enzyme systems described by Jencks are a challenge for structural biology. The power of protein engineering in elucidating enzyme structure and mechanism becomes obvious in a number of contributions describing results on various systems. Knowles showed that an enzyme may have evolved to a perfect catalyst where a change either has no effect or lessens its performance. He and Ferscht demonstrated that the effects of mutation on structure and function are often predictable, and thus make it possible to derive this residue's contribution quantitatively. But there are also unexpected effects, probably due to induced structural changes, which highlight our lack of understanding of protein structure. Similar results were obtained with the other systems discussed: proteases and their inhibitors (Christiansen, Bode) hemoglobin (Nagai), dihydrofolate

6 VII reductase (Kraut), and phage lysozyme (Alber). It is obvious that the "unexplained" results are particularly challenging and their analysis may lead to new concepts. Protein engineering is a further dimension in the study of protein structure and function, and provides insights which cannot be obtained by other methods. It is clear that the generation and analysis of mutants will play a decisive role in the goal of understanding structure and function of proteins in quantitative terms, should this ever be reached. The scientific organizers had the privilege of assembling the program for an interesting theme, attractive to the speakers. The result obviously appeared interesting to many, as it attracted the largest audience that had ever attended a Mosbach Colloquium. We hope it did more than satisfy the curiosity on this rapidly developing field of protein engineering, and helped to document its potential in a realistic way. The attraction and success of a meeting rests with the speakers. We are grateful to them for corning, many from far away. They were very cooperative and made the scientific organization a pleasure. We thank the Gesellschaft fur Biologische Chemie and its President, Prof. Dr. Oesterhelt, for giving us the opportunity to organize this colloquium, and Dr. Truscheit and Dr. Gibian and their coworkers and the local authorities, who had the burden of the technical organization of this meeting. The financial support by the Deutsche Forschungsgemeinschaft and the generous sponsorship of a number of private companies is gratefully acknowledged. E.-L. Winnacker R. Huber

7 Contents How Does ATP Make Work? W.P. Jencks Hydrophobic Interactions in Proteins P.L. Privalov (With 11 Figures) Is There a Code for Protein Folding? R. Jaenicke (With 7 Figures) The Method of Protein Structure Determination by NMRin Solution: Initial New Insights Relating to Molecular Mobility K. WUthrich (With 4 Figures) A Calculated Conformation for the Folding Transition State of Bovine Pancreatic Trypsin Inhibitor M. Levitt (With 1 Figure) Conformational Entropy and Protein Stability K.A. Dill and D.O.V. Alonso (With 1 Figure) The Improvement of Catalytic Effectiveness of an Enzyme: Pseudorevertant Triosephosphate Isomerases Obtained by Random Mutagenesis of Catalytically Sluggish Mutants S.C. Blacklow, J.D.Hermes and J.R. Knowles (With 4 Figures) 59 Reaction Coordinate Approach to the Binding of Ligands to Carboxypeptidase A D.W. Christianson and W.N. Lipscomb (With 11 Figures) The Specific Interaction of Human Leukocyte Elastase with Various Protein Inhibitors W. Bode (With 7 Figures) The Interplay Between Enzyme Mechanism, Protein Structure, and Inhibitor and Catalyst Design P.A. Bartlett (With 4 Figures) Evolution of Hemoglobin as Studied by Protein Engineering and X-Ray Crystallography D. Altschuh, J. Tame and K. Nagai (With 5 Figures) Design and Construction of Biologically Active Peptides and Proteins Including Enzymes E.T. Kaiser 109 Computer Modeling with a Protein Data Base T.A. Jones

8 x Automated and High-Speed DNA Sequencing - Computer Technology Promotes Biological Advances A. Wada (Hith 2 Figures) Synthetic Antibodies with a Known Three-Dimensional Structure A. Pllickthun, A. Skerra, R. Glockshuber and J. Stadlmliller (With 3 Figures)

9 Contributors You will find the addresses at the beginning of the respective contribution Alonso, D.O.V. 51 Altschuh, D. 96 Bartlett, P.A. 86 Blacklow, S.C. 59 Bode, W. 75 Christianson, D.W. 65 Dill, K.A. 51 Glockshuber, R. 123 Hermes, J.D. 59 Jaenicke, R. 16 Jencks, W.P. 1 Jones, T.A. 113 Kaiser, E.T. 109 Knowles, J.R. 59 Levitt, M. 45 Lipscomb, W.N. 65 Nagai, K. 96 P1Uckthun, A. 123 Privalov, P.L. 6 Skerra, A. 123 StadlmUller, J. 123 Tame, J. 96 Wada, A. 116 WUthrich, K. 37