Membrane Proteins Production for Structural Analysis

Isabelle Mus-VeteauEditor

Membrane Proteins Production for Structural Analysis

1 3

ISBN 978-1-4939-0661-1 ISBN 978-1-4939-0662-8 (eBook)DOI 10.1007/978-1-4939-0662-8Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014940924

© Springer Science+Business Media New York 2014This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, 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. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

EditorIsabelle Mus-VeteauInstitute of Molecular and Cellular Pharmacology, UMR-CNRS 7275,University of Nice-Sophia Antipolis Valbonne, France

v

Foreword

Membrane proteins are involved in fundamental biological processes like ion, wa-ter, or solute transport, sensing changes in the cellular environment, signal transduc-tion, and control of cell-cell contacts required to maintain cellular homeostasis and to ensure coordinated cellular activity in all organisms. Because of the importance of these proteins to living cells, their dysfunctions are responsible for numerous pathologies like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hy-pertension, and Alzheimer diseases. However, studies on these and other disorders are hampered by a lack of information about the involved proteins. Knowing the structure of these proteins and understanding their molecular mechanism is not only of fundamental biological interest, but also holds great potential for enhancing hu-man health. This is of paramount importance in the pharmaceutical industry, which produces many drugs that bind to membrane proteins and which recognizes the po-tential of many recently identified G-protein-coupled receptors (GPCRs), ion chan-nels, and transporters, as targets for future drugs.

Fifty percent of all drug targets are GPCRs, which is one of the largest and most diverse membrane protein families. Whereas high-resolution structures are available for a myriad of soluble proteins (more than 42,000 in the Protein Data Bank), atomic structures have so far been obtained for only 424 membrane pro-teins. Remarkably, this number is growing exponentially with 100 new structures determined in the last two years. However, only ten percent of membrane protein structures are derived from vertebrates. Indeed, the majority of medically and phar-maceutically relevant mammalian membrane proteins are present in tissues at very low concentration, making production of recombinant proteins in heterologous sys-tems suitable for large-scale production a prerequisite for structural studies. For the majority of mammalian membrane proteins, the production of soluble, stable and correctly folded protein is challenging. The breakthrough occurred in 2005 with the two first atomic structures of recombinant mammalian membrane proteins ob-tained from proteins overexpressed in yeast: the calcium ATPase from sarcoplasmic reticulum SERCA1A and the Kv1.2 voltage-gated potassium channel. Since then, extensive optimization of heterologous expression systems, stabilization tools, and structural analysis methods has begun to bear fruit, and the structure of 37 recombi-nant mammalian membrane proteins have been solved.

vi

With this book, we compiled some advances in heterologous expression systems, stabilization tools and structural methods that contributed to the growing number of recombinant integral membrane protein structures solved these in the past few years.

It will also facilitate the structural analysis of many other membrane proteins. I want to thank the authors of each chapter for their contribution to this book. I want to thank the authors of each chapter for their contribution to this book, which should be of strong interest for people who wish to produce membrane proteins for structural analysis.

Isabelle Mus-Veteau

Foreword

vii

Preface

Structural biology of integral membrane proteins has been in the limelight ever since the first 7 Å resolution three-dimensional structure of bacteriorhodopsin was determined by electron crystallography and published in 1975. Since then, there have been incredible advances in our ability to express any membrane protein in heterologous expression systems and purify them in a functional form suitable for crystallization. Some membrane proteins have proven to be amenable for struc-tural analysis. We now have a wealth of information on the structure and function of bacterial ion channels, transporters, respiratory complexes, and photosynthetic assemblies, which has led to the award of a number of Nobel Prizes, highlight-ing the importance of these proteins in biology and the difficulty in determining their structures. Nevertheless, structure determination of mammalian membrane proteins has proven much more difficult, but in the last five years there have been dramatic advances in our understanding of why these proteins are more difficult than their bacterial counterparts. This has been demonstrated most graphically with the structure determination of G-protein-coupled receptors (GPCRs), where a series of complementary and generic engineering and crystallization methodologies have been developed in different laboratories around the world, making it possible to determine the structure of any GPCR provided that enough authentically folded receptors can be expressed.

Expression of many integral membrane proteins remains challenging. Hu-man membrane proteins often require molecular chaperones to fold correctly in a process that may take hours, and the proteins may be far less stable than their bacterial homologues. Thus the challenge of producing milligrams of correctly folded protein remains. This volume addresses many of the problems associated with producing membrane proteins and more importantly how to purify them in a functional form using stabilizing detergents and detergent mimetics, allowing sub-sequent biophysical and structural analyses. Every membrane protein behaves in its own unique fashion, with quirks and peccadilloes enough to make each protein a challenge to express, purify, and crystallize. Thus the more tools we have in our toolbox of protocols for handling membrane proteins, the greater chance we have of making even the most wayward membrane protein behave.

viii Preface

Structural biology of membrane proteins is entering a new era. Electron cryo-microscopy was recently used for the first structure determination of an integral membrane protein to 3.4 Å resolution by single particle analysis; in the next few years as technology develops, this will become easier and promises the possibility of determining structures of any protein over about 250 kDa in size without the need for crystallization. The X-ray free electron laser has shown how high-resolu-tion structures can be determined from micron-sized crystals of membrane proteins using only a few hundred micrograms of purified protein. New developments in electron diffraction of sub-micron crystals also show great promise for future struc-tural analyses. Structure-based drug design for GPCRs is a reality, with multiple structures being determined of a single receptor bound to different drug candidates. However, if you cannot express and purify your membrane protein of interest in a biologically relevant state, then these great advances are superfluous. Thus, there will always be the need for improvements in expression systems and for careful biochemical analysis of the proteins produced.

Christopher G. Tate16th January 2014

ix

Contents

1 Membrane Protein Production for Structural Analysis ......................... 1Isabelle Mus-Veteau, Pascal Demange and Francesca Zito

2 Membrane Protein Quality Control in Cell-Free Expression Systems: Tools, Strategies and Case Studies............................................ 45Davide Proverbio, Erik Henrich, Erika Orbán, Volker Dötsch and Frank Bernhard

3 Bacterial Expression and Stabilization of GPCRs .................................. 71Jean-Louis Banères

4 Membrane Protein Production in Escherichia coli: Overview and Protocols .............................................................................................. 87Georges Hattab, Annabelle Y. T. Suisse, Oana Ilioaia, Marina Casiraghi, Manuela Dezi, Xavier L. Warnet, Dror E. Warschawski, Karine Moncoq, Manuela Zoonens and Bruno Miroux

5 Lactococcus lactis: Recent Developments in Functional Expression of Membrane Proteins ........................................................... 107Sana Bakari, François André, Daphné Seigneurin-Berny, Marcel Delaforge, Norbert Rolland and Annie Frelet-Barrand

6 Overexpression of Membrane Proteins in Saccharomyces cerevisiae for Structural and Functional Studies: A Focus on the Rabbit Ca2+-ATPase Serca1a and on the Yeast Lipid “Flippase” Complex Drs2p/Cdc50p ......................................................... 133Cédric Montigny, Hassina Azouaoui, Aurore Jacquot, Marc le Maire, Christine Jaxel, Philippe Champeil and Guillaume Lenoir

7 Amphipols: A General Introduction and Some Protocols ...................... 173Manuela Zoonens, Francesca Zito, Karen L. Martinez and Jean-Luc Popot

x Contents

8 New Amphiphiles to Handle Membrane Proteins: “Ménage à Trois” Between Chemistry, Physical Chemistry, and Biochemistry ..................................................................................... 205Grégory Durand, Maher Abla, Christine Ebel and Cécile Breyton

9 Building Model Membranes with Lipids and Proteins: Dangers and Challenges .......................................................................... 253James N. Sturgis

10 Analytical Ultracentrifugation and Size-Exclusion Chromatography Coupled with Light Scattering for the Characterization of Membrane Proteins in Solution ............... 267Aline Le Roy, Cécile Breyton and Christine Ebel

11 Lipidic Cubic Phase Technologies for Structural Studies of Membrane Proteins ............................................................................. 289Andrii Ishchenko, Enrique Abola and Vadim Cherezov

12 Micelles, Bicelles, Amphipols, Nanodiscs, Liposomes, or Intact Cells: The Hitchhiker’s Guide to the Study of Membrane Proteins by NMR ............................................................. 315Laurent J. Catoire, Xavier L. Warnet and Dror E. Warschawski

13 Foundations of Biomolecular Simulations: A Critical Introduction to Homology Modeling, Molecular Dynamics Simulations, and Free Energy Calculations of Membrane Proteins ..................................................................................................... 347Jérôme Hénin, Marc Baaden and Antoine Taly

14 Structural Studies of TSPO, a Mitochondrial Membrane Protein ....................................................................................................... 393 Jean-Jacques Lacapere, Soria Iatmanen-Harbi, Lucile Senicourt, Olivier Lequin, Piotr Tekely, Rudra N. Purusottam, Petra Hellwig, Sebastien Kriegel, Stephanie Ravaud, Céline Juillan-Binard, Eva Pebay Peyroula and Vassilios Papadopoulos

Erratum ........................................................................................................... E1

Index .................................................................................................................. 423

xi

Contributors

M. Abla Avignon University, Avignon, France

E. Abola Department of Integrated Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

F. André Oxidative Stress and Detoxification Laboratory, UMR-CNRS 8221, Institute of Biology and Technology of Saclay, SB2SM, and Centre for Nuclear Studies and Université Paris-Suds, Gif-sur- Yvette, France

H. Azouaoui Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

M. Baaden Laboratory of Theoretical Biochemistry, Institute of Physico-Chemical Biology, French National Centre for Scientific Research, Université Paris Diderot, Paris, France

S. Bakari Oxidative Stress and Detoxification Laboratory, UMR-CNRS 8221, Institute of Biology and Technology of Saclay, SB2SM, and Centre for Nuclear Studies and Université Paris-Suds, Gif-sur-Yvette, France

J.-L. Banères Institute of Biomolecules Max Mousseron, UMR-CNRS 5247, Faculty of Pharmacy, Université Montpellier 1 and 2, Montpellier, France

Frank Bernhard Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

C. Breyton Institute of Structural Biology, French National Centre for Scientific Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

M. Casiraghi Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

xii Contributors

L. J. Catoire Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

P. Champeil Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

V. Cherezov Department of Integrated Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

M. Delaforge Oxidative Stress and Detoxification Laboratory, UMR-CNRS 8221, Institute of Biology and Technology of Saclay, SB2SM, and Centre for Nuclear Studies and Université Paris-Suds, Gif-sur-Yvette, France

P. Demange Institute of Pharmacology and Structural Biology, UMR-CNRS 5089, Université de Toulouse, Toulouse, France

M. Dezi Laboratory of Crystallography and NMR Biology, UMR-CNRS 8015, Université Paris Descartes, Paris, France

V. Dötsch Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

G. Durand Avignon University, Avignon, FranceInstitute of Biomolecules Max Mousseron, UMR-CNRS 5247, Montpellier, France

C. Ebel Institute of Structural Biology, French National Centre for Scientific Research, Centre for Nuclear Studies, and Université Grenoble Alpes, Grenoble, France

A. Frelet-Barrand Oxidative Stress and Detoxification Laboratory, UMR-CNRS 8221, Institute of Biology and Technology of Saclay, SB2SM, and Centre for Nuclear Studies and Université Paris-Suds, Gif-sur-Yvette, France

G. Hattab Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, Université Paris Diderot, Paris, France

P. Hellwig Laboratory of Vibrational Spectroscopy and Electrochemistry of Biomolecules, UMR-CNRS 7177, Université de Strasbourg, Strasbourg, France

J. Hénin Laboratory of Theoretical Biochemistry, Institute of Physico-Chemical Biology, French National Centre for Scientific Research, Université Paris Diderot, Paris, France

E. Henrich Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

xiiiContributors

S. Iatmanen-Harbi BioMolecules Laboratory, UMR-CNRS 7203, Université Pierre et Marie Curie and Ecole Normale Supérieure, Paris, France

O. Ilioaia Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

A. Ishchenko Department of Integrated Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

A. Jacquot Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

C. Jaxel Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

C. Juillan-Binard Institute of Structural Biology, French National Centre for Scientific Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

S. Kriegel Laboratory of Vibrational Spectroscopy and Electrochemistry of Biomolecules, UMR-CNRS 7177, Université de Strasbourg, Strasbourg, France

J.-J. Lacapere BioMolecules Laboratory, UMR-CNRS 7203, Université Pierre et Marie Curie and Ecole Normale Supérieure Paris, France

M. le Maire Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

A. Le Roy Institute of Structural Biology and The European Molecular Biology Laboratory, Integrated Structural Biology Grenoble, French National Centre for Scientific Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

G. Lenoir Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

O. Lequin BioMolecules Laboratory, UMR-CNRS 7203, Ecole Normale Supérieure, Université Pierre et Marie Curie, Paris, France

K. L. Martinez Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark

B. Miroux Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

xiv Contributors

K. Moncoq Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

C. Montigny Laboratory of Membrane Proteins, Institute of Biology and Technology of Saclay, UMR-CNRS 8221, Centre for Nuclear Studies and Université Paris-Sud, Gif-sur-Yvette, France

I. Mus-Veteau Institute for Molecular and Cellular Pharmacology, UMR-CNRS 7275, University of Nice-Sophia Antipolis, Valbonne, France

E. Orbán Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

V. Papadopoulos The Research Institute of the McGill University Health Center, Department of Medicine, McGill University, Montreal, QC, Canada

E. Pebay-Peyroula Institute of Structural Biology, French National Centre for Scientific Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

J.-L. Popot Institute of Physico-Chemical Biology, UMR-CNRS 7099, Université Paris Diderot, Paris, France

D. Proverbio Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany

R. N. Purusottam BioMolecules Laboratory, UMR-CNRS 7203, Université Pierre et Marie Curie and Ecole Normale Supérieure, Paris, France

S. Ravaud Institute of Structural Biology, French National Centre for Scientific Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

N. Rolland Cell & Plant Physiology Laboratory, Institute of Sciences Research and Technologies, UMR-CNRS 5168, National Institute of Agronomical Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

D. Seigneurin-Berny Cell & Plant Physiology Laboratory, Institute of Sciences Research and Technologies, UMR-CNRS 5168, National Institute of Agronomical Research, Centre for Nuclear Studies, Université Grenoble Alpes, Grenoble, France

L. Senicourt BioMolecules Laboratory, UMR-CNRS 7203, Université Pierre et Marie Curie and Ecole Normale Supérieure Paris, France

J. N. Sturgis Engineering Laboratory of Macromolecular Systems, UMR-CNRS 7255 and Aix-Marseille University, Marseille, France

xvContributors

A. Y. T. Suisse Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

A. Taly Laboratory of Theoretical Biochemistry, Institute of Physico-Chemical Biology, French National Centre for Scientific Research, Université Paris Diderot, Paris, France

P. Tekely BioMolecules Laboratory, UMR-CNRS 7203, Université Pierre et Marie Curie and Ecole Normale Supérieure, Paris, France

X. L. Warnet Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

D. E. Warschawski Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

F. Zito Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

M. Zoonens Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099, Institute of Physico-Chemical Biology, and Université Paris Diderot, Paris, France

xvii

List of Abbreviations

3D three-dimensionalAA amino acidsABC ATP-binding cassetteABF adaptive biasing forceAch acetylcholineAChBP acetylcholine binding proteinADP adenosine diphosphateAMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacidAPO 10, dimethyldecylphosphine oxideAPol amphipathic polymersAPol amphipolATD amino-terminal domainATP adenosine triphosphateAUC analytical ultracentrifugationBAD biotin acceptor domainBCA bicinchoninic acidBR bacteriorhodopsinBrij35 polyoxyethylene-(23)-lauryl-etherBrij58 polyoxyethylene-(20)-cetyl-etherBrij72 polyoxyethylene-(2)-stearyl-etherBrij78 polyoxyethylene-(20)-stearyl-etherBrij98 polyoxyethylene-(20)-oleyl-etherBz-ATP benzoyl-benzoyl-ATPC12E7 heptaethylene glycol monododecyl etherC12E8 octaethylene glycol monododecyl etherCAC critical aggregation concentrationCD circular dichroismCDM chemically defined mediumcDNA complementary DNACF cell freeCFE cell-free expressionCHAPS 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonat

xviii List of Abbreviations

CHAPSO cholamidopropyl-dimethylammonio-hydroxy-propanesulfonateCHS cholesteryl hemisuccinateCMC critical micellar concentrationCPP critical packing parameterCXCR chemokine receptorCYMAL-5 5-cyclohexyl-1-pentyl-β-D-maltosideCymal6 6-cyclohexyl-1-hexyl-β-D-maltosideDAG diacylglycerolDAPol partially deuterated A8-35 amphipolsDDM n-dodecyl-β-D-maltopyranosideDHPC 1,2-diheptanoyl-sn-glycero-3-phosphocholinediC7PC 1,2-diheptanoyl-sn-glycero-3-phosphocholineDigit digitoninDLS dynamic light scatteringDM n-decyl-β-D-maltopyranosideDMPA 1,2-ditetradecanoyl-sn-glycero-3-phosphateDMPC dimyristoyl phosphatidylcholineDMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholineDMPE 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamineDMPG 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)DNA deoxyribonucleic acidDNP dynamic nuclear polarizationDOPA 1,2-dioleoyl-sn-glycero-3-phosphateDOPC 1,2-dioleoyl-sn-glycero-3-phosphocholineDOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineDOPG 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)DPC n-dodecylphosphocholineC12-PC n-dodecylphosphocholineDTPC ditridecanoyl phosphatidylcholineE. coli Escherichia coliECD extracellular domainECF energy coupling factorEM electron microscopyEPR electronic paramagnetic resonanceER endoplasmic reticulumF6-DigluM N-1,1-di[(O-β-D-glucopyranosyl)oxymethyl]ethyl-4-thia-

7,7,8,8,9,9,10,10,11,11,12,12,12-tridecafluorododecanamideFAPol fluorescently-labelled amphipolFC-12 FOS choline 12 or N-dodecylphosphocholineFos14 n-tetradecylphosphocholineFos16 n-hexadecylphosphocholineFS fluorinated surfactantsF-surfactant fluorinated surfactantGABA gamma aminobutyric acidGFP green fluorescent protein

xixList of Abbreviations

GpA glycophorin AGPCR G protein-coupled receptorGST glutathione S-transferaseHDM n-hexadecyl-β-D-maltopyranosideHMG-CoA 3-hydroxy-3-methylglutaryl coenzyme AIB inclusion bodyIEC ion exchange chromatographyiGluR ionotropic glutamate receptorIMAC immobilized-metal affinity chromatographyIMP integral membrane proteinIMV inner membrane vesiclesINV inverted vesiclesIPTG isopropylβ-D-1-thiogalactopyranosidekDa kilodaltonsLAB lactic acid bacteriaLBD ligand-binding domainLCP lipidic cubic phaseLDAO N,N-dimethyldodecylamine N-oxideC12-DAO N,N-dimethyldodecylamine N-oxideLDAO lauryldimethylamine oxideLGIC ligand-gated ion channelLIC ligation independent cloningLMPC 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac(1-choline)]LMPG 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]LPC L-α-lysophosphatidylcholineLPPG 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]LPS lipo-polysaccharidesLS n-lauroyl sarcosineMAG mono acyl glycerolMALS multi-angle laser light scatteringMBP maltose binding proteinMCD methyl-b-D-cyclodextrinMCS multi-cloning siteMD molecular dynamicsMFS major facilitator superfamilyPBSA Poisson Boltzmann surface accessibilityMNG maltose-neopentyl glycolMOMP major outer membrane proteinMP membrane proteinsMR molecular replacementMS microsomesMSP membrane scaffold proteinnAChR nicotinic acetylcholine receptorNADPH nicotinamide adenine dinucleotide phosphate reducedNApol neutral amphipathic polymers

xx List of Abbreviations

NaPol non-ionic amphipolsNBD nucleotide binding domainNCS-ATP 8-thiocyano-ATPNG n-nonyl-β-d-glucopyranosideNICE NIsin controlled gene expressionNi-NTA Ni2+-nitrilotriacetic acidNMA normal mode analysisNMDA N-methyl-D-aspartateNMR nuclear magnetic resonancenOG n-octyl-β-D-glucopyranosideNvoy NV10 polymerOG octyl-β-D-glucopyranosideOGorC8-G n-octyl-β-D-glucosideOGCP oxoglutarate carrier proteinOMV outer membrane vesiclesONGPG octyl glucose neopentyl glycolOTG n-octyl-ß-D-glucopyranosideP2X ATP-gated receptorPC L-α-phosphatidylcholinePE,L-α-phosphatidylethanolaminePC-APol phosphocholine-based amphipolPCC-α-M propylcyclohexylcyclohexyl-α-D-maltosidePDB Protein Data BankPDC protein detergent complexPE phosphatidylethanolamineperDAPol perdeuterated A8-35 amphipolsPL polar lipid extractpLGIC pentameric ligand-gated ion channelPMF potential of mean forcePOPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinePOPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolaminePOPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)PS phosphatidylserineRDC residual dipolar couplingRI refractive indexRNA ribonucleic acidRS Stokes radiusSANS small-angle neutron scatteringSAPol sulfonated amphipolSDS sodium dodecyl sulfateSDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresisSEC size exclusion chromatographySEC/MALS size exclusion chromatography coupled to static and dynamic light

scattering, absorbance and refractive index detectionsSERCA sarco/endoplasmic reticulum Ca2+-ATPaseSF selectivity filter

xxiList of Abbreviations

SFX serial femtosecond crystallographySMD steered molecular dynamicsSPR surface plasmon resonancessNMR solid state NMRSV sedimentation velocityT4L T4 lysozymeTEM transmission electronic microscopyTEV tobacco etch virusTGN trans-Golgi networkTL total lipid extractTM transmembraneTMP total membrane proteinsTMRM tetramethyl-rhodamine maleimideTNP trinitrophenyltOmpA transmembrane domain of the outer membrane protein A from

Escherichia coliTRIS tris(hydroxylmethyl)amidomethaneTROSY transverse relaxation optimized spectroscopyTrp tryptophanTRX thioredoxinTX-100 Triton X-100UDM undecyl-β-D-maltosideUV ultravioletVBEx vector backbone exchangeWALP tryptophan, alanine and leucine peptideWT wild typeXFEL X-ray free-electron laserZIREX zinc regulated expression systemβ-DDMor n-dodecyl-β-D-maltosideC12-M

xxiii

About the Editor

Dr. Isabelle Mus-Veteau is a biochemist and biophysicist specialist in membrane protein characterization. She obtained her PhD in microbiology and cell biology at the University of Marseille in France. She has held a French National Centre for Scientific Research (CNRS) tenure position at the Institute of Molecular and Cellular Pharmacology (IPMC, Sophia Antipolis near Nice, France), where she su-pervises projects on the characterization of the Hedgehog receptor Patched. She was a member of the CNRS tenure position recruitment committee and is currently a member of the executive committees of the French Biophysical Society and of the Membrane Group Society. She organized two international summer schools on membrane protein production for structural analysis and two international con-gresses on membrane biophysics.