Protein Engineering Handbook, Volume 1 & Volume 2

992 Pages·2009·13.889 MB·English

Protein Engineering
Handbook
Volume 1
Edited by
Stefan Lutz and
Uwe T. Bornscheuer
Protein Engineering
Handbook
Volume 2
Edited by
Stefan Lutz and
Uwe T. Bornscheuer
Further Reading
Cox, M. M., Phillips, G. N. (eds.)
Handbook of Proteins
Structure, Function and Methods. 2 Volume Set
2008
Hardcover
ISBN: 978-0-470-06098-8
Miller, L. W. (eds.)
Probes and Tags to Study Biomolecular Function
2008
Hardcover
ISBN: 978-3-527-31566-6
Lengauer, T. (ed.)
Bioinformatics – From Genomes to Therapies
2007
Hardcover
ISBN: 978-3-527-31278-8
Schreiber, S. L., Kapoor, T., Wess, G. (eds.)
Chemical Biology
From Small Molecules to Systems Biology and Drug Design
2007
Hardcover
ISBN: 978-0-470-84984-2
Aehle, W. (ed.)
Enzymes in Industry
Production and Applications
2007
Hardcover
ISBN: 978-3-527-31689-2
Meyers, R. A. (ed.)
Proteins
From Analytics to Structural Genomics
2006
Hardcover
ISBN: 978-3-527-31608-3
Protein Engineering Handbook
Volume 1
Edited by
Stefan Lutz and Uwe T. Bornscheuer
Protein Engineering Handbook
Volume 2
Edited by
Stefan Lutz and Uwe T. Bornscheuer
The Editors All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
Prof. Dr. Stefan Lutz publisher do not warrant the information
Dept. of Chemistry contained in these books, including this book, to
Emory University be free of errors. Readers are advised to keep in
1515 Dickey Drive mind that statements, data, illustrations,
Atlanta GA 30322 procedural details or other items may
USA inadvertently be inaccurate.
Prof. Dr. Uwe T. Bornscheuer Library of Congress Card No.: applied for
Dept. of Biotechnology and Enzyme Catalysis
Institute of Biochemistry British Library Cataloguing-in-Publication Data
Greifswald University A catalogue record for this book is available from
Felix-Hausdorff-Str. 4 the British Library.
17487 Greifswald
Bibliographic information published by the
Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this
publication in the Deutsche Nationalbibliografi e;
detailed bibliographic data are available on the
Internet at <http://dnb.d-nb.de>.
© 2009 WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim
All rights reserved (including those of translation
into other languages). No part of this book may
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are not to be considered unprotected by law.
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Printed in the Federal Republic of Germany
Printed on acid-free paper
ISBN: 978-3-527-31850-6
V
Contents
Volume 1
Preface XXVII
List of Contributors XXXI
1 Guidelines for the Functional Analysis of Engineered and Mutant
Enzymes 1
Dale E. Edmondson and Giovanni Gadda
1.1 Introduction 1
1.2 Steady-State Kinetics 2
1.3 Enzyme Assays and the Acquisition of Initial Velocity Data 3
1.3.1 Biological Sample Appropriate for Assay 3
1.3.2 Enzymatic Assays 4
1.3.3 Analysis of Initial Rate Data 6
1.3.4 Determination of Functional Catalytic Site Concentrations 8
1.4 Steady-State Kinetic Parameters and Their Interpretation 8
1.4.1 pH-Dependence of Steady-State Kinetic Parameters 11
1.4.2 Analysis of Two-Substrate Enzymes 11
1.5 Concluding Remarks 12
References 12
2 Engineering Enantioselectivity in Enzyme-Catalyzed Reactions 15
Romas Kazlauskas
2.1 Introduction 15
2.2 Molecular Basis for Enantioselectivity 18
2.2.1 Enzymes Stabilize Transition States for Fast-Reacting Enantiomers
Better than Slow-Reacting Enantiomers 18
2.2.2 The Slow-Reacting Enantiomer Fits by Exchanging Two
Substituents 18
2.2.3 The Slow Enantiomer Fits by an Umbrella-Like
Inversion 19
2.3 Qualitative Predictions of Enantioselectivity 23
Protein Engineering Handbook. Edited by Stefan Lutz and Uwe T. Bornscheuer
Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31850-6
VI Contents
2.3.1 Comparing Substrate Structures Leads to Empirical Rules and Box
Models 23
2.3.2 Computer Modeling Based on X-Ray Structures of Enzymes 25
2.3.3 What Is Missing from Current Computer Modeling? 26
2.4 Protein Engineering to Increase or Reverse Enantioselectivity 30
2.4.1 Mutations Closer to the Active Site Increase Enantioselectivity More
Effectively than Mutations Far from the Active Site 30
2.4.2 Reversing Enantioselectivity by Exchanging Locations of Binding Sites
or a Catalytic Group 36
2.5 Concluding Remarks 40
References 41
3 Mechanism and Catalytic Promiscuity: Emerging Mechanistic
Principles for Identifi cation and Manipulation of Catalytically
Promiscuous Enzymes 47
Stefanie Jonas and Florian Hollfelder
3.1 Introduction 47
3.2 Calculation of Rate Accelerations 52
3.3 Catalytic Features and Their Propensity for Promiscuity 55
3.3.1 Metal Ions 55
3.3.2 Recognition of Transition State Charges: Analysis of the Nature of the
Transition State 61
3.3.3 Catalytic Dyads and Triads 63
3.3.4 General Acid/Base Catalysts in Promiscuous Functional Motifs in
Catalytic Superfamilies 64
3.4 Steric Effects and Structural Constriction in the Active Site: Product
Promiscuity 67
3.5 Medium Effects in Enzyme Active Sites 70
3.6 Conclusions 71
References 72
4 Φ-Value Analysis of Protein Folding Transition States 81
Neil Ferguson and Alan R. Fersht
4.1 Introduction 81
4.2 Theoretical Principles of Protein Engineering 82
4.2.1 Overview 82
4.2.2 Basic Concepts 83
4.2.3 Theory of Φ-Value Analysis 87
4.2.4 Relationship between Φ and Leffl er α 90
4.2.5 Linear Free-Energy Relationships and Denaturant Concentration 93
4.3 Guidelines for the Determination of Accurate Φ-Values 95
4.3.1 Buffer Preparation and Selection 96
4.3.2 Optimization of Experimental Conditions 97
4.3.3 Equilibrium Denaturation Experiments 99
4.3.3.1 Practical Considerations 99
Contents VII
4.3.3.2 Curve-Fitting 103
4.3.4 Kinetic Measurements 105
4.3.4.1 Practical Considerations 107
4.3.4.2 Curve Fitting 110
4.3.4.3 Error Analysis for Chevron Plots 113
4.4 Conclusions 115
Acknowledgments 116
References 116
5 Protein Folding and Solubility: Pathways and High-Throughput
Assays 121
Adam C. Fisher, Thomas J. Mansell, and Matthew P. DeLisa
5.1 Introduction 121
5.2 Biosynthesis of Natural Proteins in Bacteria 122
5.2.1 Recombinant Protein Folding 122
5.2.2 Protein Misfolding and Inclusion Body Formation 123
5.2.3 Proteolysis 124
5.2.4 Cytoplasmic Chaperones 124
5.2.5 Export Pathways 125
5.3 Biosynthesis of de novo-Designed Proteins in Bacteria 126
5.4 Combinatorial Strategies for Assaying Protein Folding in
Bacteria 126
5.4.1 Initial Protein-Folding Studies 128
5.4.2 Protein Chimeras 128
5.4.3 Split Proteins 129
5.4.4 Genetic Response 130
5.4.5 Cellular Quality Control Systems 130
5.5 Structural Genomics 131
5.6 Protein-Misfolding Diseases 132
5.7 Future Directions 135
5.7.1 Folding versus Solubility 137
References 138
6 Protein Dynamics and the Evolution of Novel Protein Function 147
Jörg Zimmermann, Megan C. Thielges, Wayne Yu and Floyd E. Romesberg
6.1 Introduction 147
6.2 Physical Background 149
6.2.1 Flexibility, Conformational Heterogeneity and Time Scales of Protein
Dynamics 149
6.2.2 Protein Dynamics and Thermodynamics of Molecular
Recognition 151
6.3 Experimental Studies of Protein Dynamics 153
6.3.1 NMR Relaxation Experiments 153
6.3.2 Ultrafast Laser Spectroscopy 154
6.4 Experimental Techniques 158
VIII Contents
6.4.1 Time-Correlation Function and the Spectral Density of Protein
Motions 158
6.4.2 NMR Relaxation Techniques to Determine ρ(ω) 160
6.4.3 Ultrafast Laser Spectroscopy to Determine C(t) and ρ(ω) 160
6.4.4 Additional Approaches to the Characterization of Protein
Dynamics 162
6.4.5 Chromophores to Probe Protein Dynamics 164
6.5 Case Study: Protein Dynamics and the Evolution of Molecular
Recognition within the Immune System 165
6.6 Implications for Protein Engineering 172
References 173
7 Gaining Insight into Enzyme Function through Correlation with Protein
Motions 187
Nicolas Doucet and Joelle N. Pelletier
7.1 Introduction 187
7.1.1 Enzyme Catalysis – the Origin of Rate Acceleration 187
7.1.2 Proteins Are Intrinsically Dynamic Molecules 188
7.1.3 Are Protein Motions Essential in Promoting the Catalytic Step of
Enzyme Reactions? 190
7.2 Experimental Investigation of Enzyme Dynamics during
Catalysis 191
7.2.1 Quantum Tunneling Revealed by Unusually Large Kinetic Isotope
Effects (KIEs): Are Enzyme Dynamics Involved? 191
7.2.1.1 Varying Atomic Mass Can Alter the Rate of Proton Transfer 192
7.2.1.2 KIEs Reveal Quantum Tunneling 192
7.2.1.3 Quantum Tunneling and Protein Dynamics 192
7.2.2 Nuclear Magnetic Resonance: Experimental Observation of Protein
Dynamics over a Broad Range of Time Scales 193
7.2.2.1 Extracting Information on Protein Dynamics by NMR 194
7.2.2.2 NMR Dynamics of Enzymes 194
7.2.3 Crystallographic Evidence of Motions in Enzymes 197
7.2.3.1 Time-Resolved X-Ray Crystallography 197
7.2.3.2 Motional Behavior in the Course of Enzyme Action 198
7.2.4 Computational Methods 199
7.2.4.1 Molecular Dynamics Simulations: Computational Models of Protein
Motions 199
7.2.4.2 Combining Quantum Mechanics with Molecular Mechanics:
QM/MM 200
7.3 Future Challenges 201
7.3.1 Promising New Methodologies for the Study of Enzyme
Dynamics 201
7.3.2 NMR: Improving Methodologies 202
7.3.3 Kinetic Crystallography: Snapshots of a Protein in Various
States 203