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MEMBRANE STRUCTURAL BIOLOGY WITH BIOCHEMICAL AND BIOPHYSICAL FOUNDATIONS

Second Edition

This textbook provides a strong foundation and a clear overview for students of membrane biology and an invaluable synthesis of cutting-edge research for working scientists. The text retains its clear and engaging style, providing a solid background in membrane biochemistry, while also incorporating the approaches of biophysics, genetics, and cell biology to investigations of membrane structure, function, and biogenesis to provide a unique overview of this fast-moving field.

A wealth of new high-resolution structures of membrane proteins are presented, including the Na + -K + pump and a receptor G protein complex, offering exciting insights into how they function. All key tools of current membrane research are described, including detergents and model systems, bioinformatics, protein-folding methodology, crystallography and diffraction, EPR and NMR spectroscopy, and molecular modeling. This comprehensive and up-to-date text, emphasizing the correlations between membrane research and human health, provides a solid foundation for all those working in this field.

Mary Luckey is Professor Emerita in the Department of Chemistry and Biochemistry at San Francisco State University. She earned her Ph.D. in Biochemistry at the University of California Berkeley with the first identification of an iron transport protein in the bacterial outer membrane. Her postdoctoral work demonstrated the specificity of the E. coli maltoporin in proteoliposomes. While continuing research on maltoporin structure and function, she has taught biochemistry for over 25 years, including the graduate-level membrane biochemistry course that provided the impetus for this book.

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Cambridge University Press978-1-107-03063-3 - Membrane Structural Biology: With Biochemical and Biophysical Foundations: Second EditionMary LuckeyFrontmatterMore information

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MEMBRANE STRUCTURAL BIOLOGY WITH BIOCHEMICAL AND BIOPHYSICAL FOUNDATIONSSECOND EDITION

MARY LUCKEY

San Francisco State University






University Printing House, Cambridge CB2 8BS, United Kingdom

Published in the United States of America by Cambridge University Press, New York

It furthers the University’s mission by disseminating knowledge in the pursuit of education, learning and research at the highest international levels of excellence.

www.cambridge.org Information on this title: www.cambridge.org/9781107030633

© Mary Luckey 2014

This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press.

First published 2008 Reprinted 2011 (twice) Second Edition 2014

Printed in the United States by Sheridan Inc.

Library of Congress Cataloging in Publication data Luckey, Mary, 1948– Membrane structural biology : with biochemical and biophysical foundations / Mary Luckey. – Second edition. pages cm Includes bibliographical references and index. ISBN 978-1-107-03063-3 (hardback) 1. Membranes (Biology) 2. Membrane lipids. 3. Membrane proteins. I. Title. QH601.L75 2014 571.6′4–dc23 2013028009

ISBN 978-1-107-03063-3 Hardback

Additional resources for this publication at www.cambridge.org/Luckey2 Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.

Cover image:

The title page shows high-resolution structures of membrane proteins incorporated into a simulated lipid bilayer. The proteins are, from left to right, β2A in complex with an agonist and a trimeric G-protein, the heme receptor HasR in complex with the heme-binding protein HasA, the trimeric aspartate transporter GltPh that is a homolog for neurotransmitter transporters, the SecYEG translocon in complex with the energizing subunit SecA, the amino acid transporter LeuT that is another homolog for neurotransmitter transporters, the Na+, K+ ATPase, the dimeric chloride transporter ClC, and P-glycoprotein, a dimeric transporter that extrudes drugs.

Kindly provided by J. C. Gumbart, Georgia Institute of Technology, and E. Tajkhorshid, University of Illinois.






In memory of Amy L. Davidson, 1958–2013, insightful scientist,

meticulous experimentalist, gracious colleague, and good friend.






vii

CONTENTS IN BRIEF

Contents ix

Preface xiv

1 Introduction 1

2 The Diversity of Membrane Lipids 14

3 Tools for Studying Membrane Components: Detergents and Model Systems 42

4 Proteins in or at the Bilayer 69

5 Bundles and Barrels 105

6 Functions and Families 130

7 Protein Folding and Biogenesis 168

8 Diffraction and Simulation 203

9 Membrane Enzymes 226

10 Membrane Receptors 257

11 Transporters 284

12 Channels 328

13 Electron Transport and Energy Transduction 358

14 In Pursuit of Complexity 385

Appendix I: Abbreviations 393

Appendix II: Single-Letter Codes for Amino Acids 397

Index 399






ix

List of boxes xiii Preface xiv

1 INTRODUCTION 1

General features of membranes 1 Paradigm 1: the amphiphilic molecules in

membranes assemble spontaneously due to the hydrophobic effect 4

Paradigm 2: the Fluid Mosaic Model describes the membrane structure 5

A shift in the paradigm: biomembranes have lateral domains that form “rafts” 9

A view for the future: dynamic protein complexes crowd the membrane interior and extend its borders 9

For further reading 13

2 THE DIVERSITY OF MEMBRANE LIPIDS 14

The acyl chains 14 Complex lipids 17

Phospholipids 19 Sphingolipids 20 Sterols and linear isoprenoids 21

The lipid bilayer matrix 22 Structure of bilayer lipids 22 Diffusion of bilayer lipids 24 Lipid asymmetry and membrane thickness 27

Lipid polymorphism 28 Lamellar phase 28 Hexagonal phase and the amphiphile

shape hypothesis 29 Cubic phase 30

Miscibility of bilayer lipids 31 Lateral domains and lipid rafts 34

Detergent-resistant membranes 35 Diversity of lipids 37 Conclusion 39 For further reading 41

3 TOOLS FOR STUDYING MEMBRANE COMPONENTS: DETERGENTS AND MODEL SYSTEMS 42

Detergents 43 Types of detergents 43 Mechanism of detergent action 46

CONTENTS

Membrane solubilization 49 Lipid removal 51

Model membranes 51 Monolayers 52 Planar bilayers 54 Patch clamps 57 Supported bilayers 57 Liposomes from SUVs to GUVs 60

Multilamellar vesicles 60 Small unilamellar vesicles 61 Large unilamellar vesicles 61 Short-chain/long-chain unilamellar

vesicles 62 Giant unilamellar vesicles 62

Mixed micelles and bicelles 63 Blebs and blisters 64 Nanodiscs 66

Conclusion 67 For further reading 67

4 PROTEINS IN OR AT THE BILAYER 69

Classes of proteins that interact with the membrane 69

Proteins at the bilayer surface 70 Extrinsic/peripheral membrane proteins 70 Amphitropic proteins 74 Lipid-anchored proteins 74 Reversible interactions of peripheral

proteins with the lipid bilayer 77 Effects of peripheral protein binding on

membrane lipids 77 Interactions between peripheral proteins

and lipids 78 Domains involved in binding

the membrane 81 Curvature 83 Modulation of binding 84

Proteins and peptides that insert into the membrane 85 Toxins 86 Colicins 88 Peptides 88 SecA: protein acrobatics 90

Proteins embedded in the membrane 91 Monotopic proteins 91 Integral membrane proteins 91

Protein–lipid interactions 97 Hydrophobic mismatch 101

For further reading 103






Contents

x

5 BUNDLES AND BARRELS 105

Helical bundles 105 Bacteriorhodopsin 105 Photosynthetic reaction center 109

The proteins 110Lipids 112The cofactors 112Antennae 113The reaction cycle 114

β -barrels 115 Porins 117

OmpF and OmpC 118 VDAC, a mitochondrial porin 122

Specifi c porins 123 PhoE, the phosphoporin 123LamB, the maltoporin 124 Other β -barrel transporters 124

Iron receptors 125 Outer membrane secretory proteins:

not β -barrels 126 Conclusion 127 For further reading 127

6 FUNCTIONS AND FAMILIES 130

Membrane enzymes 130 Diacylglycerol kinase 132 Presenilin, an intra-membrane protease 134 P450 cytochromes 136

Transport proteins 137 Transport classifi cation system 138 Superfamilies of ATPases 139 ABC transporter superfamily 140 Group translocation 140 Symporters 142 Antiporters 142 Ion channels 143

Membrane receptors 144 Nicotinic acetylcholine receptor 144 G protein-coupled receptors 145

Bioinformatics tools for membrane protein families 146 Predicting TM segments 146 Hydrophobicity plots 148 Orientation of membrane proteins 149 The positive-inside rule 150 Inverted repeats 151 Genomic analysis of membrane proteins 153

Helix–helix interactions 162

Proteomics of membrane proteins 163 Predicting β -barrels 164 For further reading 165

7 PROTEIN FOLDING AND BIOGENESIS 168

Protein folding 169 Folding α -helical membrane proteins 170

bR folding studies 173 Folding studies of β -barrel membrane

proteins 175 Other folding studies 177 Whole protein hydrophobicity scale 178

Biogenesis of membrane proteins 178 Export from the cytoplasm 180 The translocon 187 The translocon structure 187 TM insertion 189 Biological hydrophobicity scale 191 Topogenesis in membrane proteins 195

Misfolding diseases 198 For further reading 201

8 DIFFRACTION AND SIMULATION 203

Back to the bilayer 203 Liquid crystallography 204

Liquid crystal theory 206 Joint refi nement of x-ray and neutron

diffraction data 207 Modeling the bilayer 209

Simulations of lipid bilayers 209 Molecular dynamics 209 Monte Carlo 213

Lipids observed in x-ray structures of membrane proteins 215

Crystallography of membrane proteins 219 A multidisciplinary approach 223 For further reading 225

9 MEMBRANE ENZYMES 226

Prostaglandin H 2 synthase 227 OMPLA 230 Membrane proteases 234

Omptins 234 Intramembrane proteases 235 Rhomboid protease 236 Structure of the bacterial rhomboid GlpG 237






Contents

xi

Formate dehydrogenase 239 P-type ATPases 243

Ca 2+ ATPases 243 Na + , K + ATPase 250 Other P-type ATPases 254


10 MEMBRANE RECEPTORS 257

G protein-coupled receptors 258 Rhodopsin, a light-sensitive GPCR 259

Ground state rhodopsin 260 Activated rhodopsin 261 Rhodopsin as prototype 263

Adrenergic receptors 267 β 2 AR structure 268 β 1 AR structure 270 Activated β 2 AR in complex with G S 270

Neurotransmitter receptors 274 Glutamate receptors: GluA2 274 Cys-loop receptors and GluCl 278


11 TRANSPORTERS 284

Secondary transporters 284 MFS transporters 285

LacY, a scrutinized symporter 285 GlpT, an MFS antiporter 288 EmrD, an MFS exporter in an occluded

conformation 289 FucP, an MFS symporter in

co conformation 290 A paradigm for MFS transporters 291

Mitochondrial ADP/ATP carrier 291 AAC structure 291

Neurotransmitter transporters 294 Glutamate transporters and GltPh 294 Neurotransmitter sodium symporters

and LeuT 296 LeuT structure 298 Transport mechanism of LeuT 300

The NSS family and the LeuT (APC-fold) superfamily 300 BetP and osmoregulated transport 303

ABC transporters and beyond 306 Maltose transporter 306

The vitamin B 12 uptake system 310 BtuCD-BtuF, an ABC transport system 311 BtuB, an outer membrane transporter

energized by TonB 313 Drug effl ux systems 316

Sav1866 and P-glycoprotein, ABC exporters 316

EmrE, an example of dual topology 318 Tripartite drug effl ux via a membrane vacuum

cleaner 320 AcrB, a peristaltic pump 321 Alternating site mechanism of AcrB 322 AcrA, a periplasmic adaptor protein 323 TolC, the channel-tunnel 323 Partners of TolC 325


12 CHANNELS 328

Aquaporins and glyceroaquaporins 329 Structure of aquaporins 330

Glyceroaquaporins: GlpF 331 Human aquaporins: AQP4 333

Potassium channels 335 KcsA structure and selectivity 336 Gating and activation 337 Voltage gating 339 Gating in human potassium channels 341

Chloride channels and the CLC family 343 ClC-ec1 344 CLC channels as “broken transporters” 346

Mechanosensitive ion channels 347 MscL 349 MscS 350 MS channel gating 351

Gap junction channels 354 Conclusion 356 For further reading 356

13 ELECTRON TRANSPORT AND ENERGY TRANSDUCTION 358

Complexes of the respiratory chain 359 Complex I 359

Conformational coupling mechanism 363 Cytochrome bc 1 365

The Q cycle 366 High-resolution structures 366






Contents

xii

Cytochrome c oxidase 371 High-resolution structures 371 Oxygen reduction 373 Proton pathways 374

F 1 F 0 -ATP synthase 375 Subunit structure and function 377

F1 Domain 377 F0 Domain 379

Regulation of the F 1 F 0 -ATPase 379 Catalytic mechanism of a rotary motor 380 Rotational catalysis 380

Conclusions 383 For further reading 383

14 IN PURSUIT OF COMPLEXITY 385

Complex formation 386 Conformational changes and

dynamics 390 For further reading 392

Appendix I: Abbreviations 393 Appendix II: Single-Letter Codes for Amino Acids 397 Index 399






xiii

LIST OF BOXES

2.1 Fluorescence techniques 26 2.2 Phase diagrams 33 3.1 Surfactants and surface tension 43 3.2 Electrophysiology 54 4.1 Binding of ligands to surfaces 80 4.2 NMR determination of membrane protein structures 95 4.3 Electron paramagnetic resonance 99 5.1 NMR determination of β -barrel membrane protein structure 119 6.1 Surface dilution effects 131 6.2 Bioinformatics basics 147 6.3 Making and testing hydrophobicity plots 150 6.4 Statistical methods for TM prediction 156 7.1 Energetics of folding and inserting a hydrophobic α -helix into the bilayer 172 7.2 Evidence for cleavable signal sequences involved in protein translocation 181 7.3 Crosslinking traces nascent peptides through the translocon into the bilayer 184 7.4 Import of mitochondrial proteins 193 8.1 X-ray and neutron scattering 205 8.2 Molecular dynamics calculations 210 8.3 Studying dynamics in solution 215 9.1 Mechanism of action of prostaglandin H 2 synthase 228 10.1 Effi ciency of light-induced signal transduction 260 12.1 Mechanosensitivity 349 13.1 A modifi ed Q cycle in cytochrome b 6 f 368






xiv

PREFACE

The fi rst edition of Membrane Structural Biology met a need for a comprehensive presentation of the explosion of information about the structure and organization of biological membranes. It also acknowledged how new techniques and whole new methodologies had changed both how we acquired knowledge of the membrane and how we viewed it. With a foundation derived from basic physical and life sciences, advances in structural biology were depicted through the molecular details of membrane components provided by sophisticated dif-fraction analysis of fl uid lipid bilayers and by high-reso-lution structures of a variety of membrane proteins. As the book moved from basic membrane biochemistry to detailed examples, it covered a wide range of material at a level appropriate for both students and scientists in the fi eld. I am gratifi ed with the responses from membrane scientists all over the world.

This new edition has been expanded to include over 20 additional membrane proteins visualized in atomic detail. Discovery of superfamilies based on the protein folds shows relationships among membrane proteins, while capture of multiple states begins to disclose mechanisms. Some new topics have been introduced, other topics updated, and yes, sadly, some interesting new fi ndings had to be left out. I hope readers will jump into the literature from the key references pro-vided to learn about the exciting new fi ndings, to study these topics in more detail, and to tackle the larger and more complex systems at the frontiers of mem-brane research.

While I am responsible for the omissions and any errors, I am indebted to many people who have been generous with their time, reviewing new parts of this edition, as well as those who did so for the fi rst edi-

tion. I was assisted when I started writing the book by my former students, Dr. Aram Krauson and Dr. Andréa Dosé. For their comments on specifi c topics in the fi rst edition I thank Professors Scott Feller, Steve White, Sam Hess, Rosemary Cornell, Ehud Landau, David Hackney, Paula Booth, Bill Plachy, and Hiroshi Nikaido. For com-ments on new sections of the second edition I thank Poul Nissen, Maike Bublitz, Satinder Singh, Merritt Maduke, Brian Kobilke, Chuck Sanders, Jörg Standfuss, Bill Cramer, Reinhard Krämer, Christine Ziegler, Shelagh Ferguson-Miller, and Eduardo Perozo. I am especially grateful to those who reviewed the entire original manu-script: Professors Lin Randall and Stanley Parsons, and my former students Shyam Bhaskaran, Marla Melnick, and Jared Matt Greenberg. Lin Randall and my former student Chris Chin also read all the new chapters for the second edition.

With much appreciation I thank Professors J. C. Gumbart and Emad Tajkhorshid for the cover fi gures. In addition I want to thank the many individual scien-tists who shared their fi gures of beautiful membrane protein structures, particularly those who prepared fi gures for this edition: Shelagh Ferguson-Miller, Rose-mary Comell, Krzyszt of Palzewski, and Tivadar Orban. For her unfl agging enthusiasm and wise editorial help, I thank Dr. Katrina Halliday. Thanks as well to my col-leagues and friends who supported my progress writing the book. Finally, I deeply appreciate the affection and encouragement I received from my family, Ariel, SAM, Ryan, donna, Kesa, Amanda, and Dana, with profound gratitude for steadfast love, patience, and support from my husband Paul.

Mary Luckey






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