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Solid-State NMRApplications in biomembrane structure

Biophysical Society–IOP Series

Committee ChairpersonLes SatinUniversity of Michigan, USA

Editorial Advisory Board MembersGeoffrey Winston AbbottUC Irvine, USA

Mibel AguilarMonash University, Australia

Cynthia CzajkowskiUniversity of Wisconsin, USA

Miriam GoodmanStanford University, USA

Jim SellersNIH, USA

Joe HowardYale University, USA

Meyer JacksonUniversity of Wisconsin, USA

Da-Neng WangNew York University, USA

Kathleen HallWashington University in St Louis, USA

David SeptUniversity of Michigan, USA

Andrea MeredithUniversity of Maryland, USA

Leslie M LoewUniversity of Connecticut School ofMedicine, USA

About the SeriesThe Biophysical Society and IOP Publishing have forged a new publishing partner-ship in biophysics, bringing the world-leading expertise and domain knowledge ofthe Biophysical Society into the rapidly developing IOP ebooks program.

The program publishes textbooks, monographs, reviews, and handbooks coveringall areas of biophysics research, applications, education, methods, computationaltools, and techniques. Subjects of the collection will include: bioenergetics; bioengin-eering; biological fluorescence; biopolymers in vivo; cryo-electron microscopy;exocytosis and endocytosis; intrinsically disordered proteins; mechanobiology; mem-brane biophysics; membrane structure and assembly; molecular biophysics; motilityand cytoskeleton; nanoscale biophysics; and permeation and transport.

Solid-State NMRApplications in biomembrane structure

Edited byFrances Separovic and Marc-Antoine Sani

School of Chemistry, Bio21 Institute, University of Melbourne,Melbourne, VIC 3010, Australia

IOP Publishing, Bristol, UK

ª IOP Publishing Ltd 2020

All rights reserved. No part of this publication may be reproduced, stored in a retrieval systemor transmitted in any form or by any means, electronic, mechanical, photocopying, recordingor otherwise, without the prior permission of the publisher, or as expressly permitted by law orunder terms agreed with the appropriate rights organization. Multiple copying is permitted inaccordance with the terms of licences issued by the Copyright Licensing Agency, the CopyrightClearance Centre and other reproduction rights organizations.

Permission to make use of IOP Publishing content other than as set out above may be soughtat permissions@ioppublishing.org.

Frances Separovic and Marc-Antoine Sani have asserted their right to be identified as the authorsof this work in accordance with sections 77 and 78 of the Copyright, Designs and Patents Act1988.

ISBN 978-0-7503-2532-5 (ebook)ISBN 978-0-7503-2530-1 (print)ISBN 978-0-7503-2533-2 (myPrint)ISBN 978-0-7503-2531-8 (mobi)

DOI 10.1088/978-0-7503-2532-5

Version: 20201201

IOP ebooks

British Library Cataloguing-in-Publication Data: A catalogue record for this book is availablefrom the British Library.

Published by IOP Publishing, wholly owned by The Institute of Physics, London

IOP Publishing, Temple Circus, Temple Way, Bristol, BS1 6HG, UK

US Office: IOP Publishing, Inc., 190 North Independence Mall West, Suite 601, Philadelphia,PA 19106, USA

Contents

Preface xiv

Foreword xv

Editor biographies xviii

List of contributors xix

1 Solid-state NMR methods for studying membrane systems 1-1Gerhard Grobner and Philip Williamson

1.1 Introduction 1-2

1.2 Using NMR as a reporter on membrane structure 1-3

1.3 Dynamics in biological membranes 1-8

1.4 Probing the surface charge of membranes with solid-state NMR 1-12

1.5 Lateral domain formation 1-14

1.6 Lipid/protein interactions 1-17

1.6.1 Insight into protein/lipid headgroup associationby 31P CP MAS NMR

1-17

1.6.2 Studying annular lipids bound to integralmembrane proteins

1-19

1.6.3 Studying non-annular lipids bound to integralmembrane proteins

1-20

1.7 Membrane remodeling 1-22

1.8 Summary 1-24

Acknowledgements 1-24

References 1-24

2 Characterization of lipid behaviour in modelbiomembranes using 2H solid-state NMR

2-1

Michael R Morrow and Jenifer L Thewalt

2.1 Introduction 2-1

2.2 The quadrupole interaction 2-2

2.3 The wideline 2H NMR spectra of chain-deuteratedphospholipids in bilayers

2-3

2.4 The quadrupole echo 2-7

2.5 Characterizing phospholipid bilayer phases usingwideline 2H NMR

2-11

v

2.6 Using spectral subtraction to identify boundariesof two-phase coexistence regions in bilayertemperature-composition phase diagrams

2-14

2.7 Beyond difference spectroscopy—defining liquid crystallinephase coexistence with 2H NMR

2-17

2.8 Summary 2-18

References 2-18

3 Deuterium solid-state NMR of whole bacteria: samplepreparation and effects of cell envelope manipulation

3-1

Valerie Booth

3.1 Structure and composition of bacterial cell envelopes 3-1

3.2 Sample preparation 3-3

3.3 Static 2H NMR of gram(−) and gram(+) bacteria 3-4

3.4 2H NMR of bacteria with cell envelope perturbation 3-7

3.5 What have we learned from 2H NMR of bacteria so far? 3-8

References 3-9

4 Solid-state NMR study of microalgal membranesand cell walls

4-1

Alexandre Poulhazan, Alexandre A Arnold, Dror E Warschawski

and Isabelle Marcotte

4.1 Overview 4-1

4.2 Microalgal cell membrane composition and architecture 4-3

4.2.1 Microalgal cell wall and plasma membrane 4-3

4.2.2 Photosynthetic organelles (thylakoids and chloroplast) 4-4

4.2.3 lipid droplets and free fatty acids 4-5

4.3 Technical considerations 4-6

4.3.1 Observable nuclei for microalgal membranes studies 4-6

4.3.2 Solution and solid-state NMR 4-7

4.4 NMR of lipid extracts and model membranes 4-9

4.5 In vivo, in-cell and in situ NMR 4-10

4.5.1 1H-NMR based approaches 4-10

4.5.2 1D 13C solid-state NMR methods 4-12

4.5.3 2D-13C and 15N-NMR experiments 4-13

4.6 Conclusion and future prospects 4-15

Acknowledgements 4-16

References 4-16

Solid-State NMR

vi

5 Determining the mechanism of action of host defensepeptides: solid-state NMR approaches

5-1

Suzana K Straus, Allen Takayesu and Prashant Kumar

5.1 Introduction 5-1

5.2 Model membranes 5-3

5.3 Whole cells 5-4

5.4 Conclusions 5-8

References 5-8

6 19F NMR of biomembranes 6-1Stephan L Grage, Sergii Afonin and Anne S Ulrich

6.1 Introduction 6-2

6.2 19F NMR 6-3

6.2.1 Advantages of 19F as NMR probe 6-3

6.2.2 Limitations of 19F NMR 6-4

6.2.3 19F NMR bridges the gap 6-5

6.2.4 Hardware requirements 6-7

6.2.5 Typical 19F NMR chemical shifts 6-8

6.3 Introducing 19F into biomembranes 6-8

6.3.1 19F-labels 6-8

6.3.2 Biosynthetic incorporation of 19F 6-12

6.3.3 Chemical introduction of 19F 6-13

6.4 Line narrowing techniques for 19F NMR of biomembranes 6-13

6.4.1 Multipulse solid-state 19F NMR 6-14

6.4.2 Oriented samples for solid-state 19F NMR 6-15

6.4.3 Magic angle spinning 19F NMR 6-17

6.4.4 Solution NMR of membrane proteins in membrane mimetics 6-17

6.5 Solid-state 19F NMR distance measurements in biomembranes 6-18

6.5.1 Distance measurements in oriented samples 6-18

6.5.2 Distance measurements under MAS 6-20

6.5.3 Multidimensional solid-state 19F NMR experiments 6-21

6.5.4 Ligands bound to membrane proteins 6-21

6.5.5 Intermolecular interactions of oligomeric membraneproteins and peptides

6-22

6.5.6 Distance measurements in solution 6-23

6.6 Solid-state 19F NMR orientation measurements in biomembranes 6-24

6.6.1 Orientation dependence in solid-state 19F NMR 6-24

6.6.2 Mobility and orientation 6-25

Solid-State NMR

vii

6.6.3 Molecular orientation in membranes from 19F NMRon oriented samples

6-25

6.6.4 Peptide re-alignment transitions in membranes revealedby solid-state 19F NMR

6-29

6.6.5 Peptide mobility in membranes 6-29

6.6.6 Orientation and mobility of lipids and smalllipid-soluble molecules

6-30

6.7 Probing the environment 6-31

6.7.1 Conformational changes 6-32

6.7.2 Probing solvent environment 6-32

6.8 19F NMR in native membranes 6-34

6.9 Conclusions 6-34

Acknowledgements 6-35

References 6-35

7 Structure, topology and dynamics of membrane-associatedpeptides by solid-state NMR

7-1

Burkhard Bechinger and Evgeniy Salnikov

7.1 Introduction 7-1

7.2 Solid-state NMR spectroscopic approaches 7-2

7.3 The anisotropy of solid-state NMR interactions 7-4

7.4 Sample preparation 7-5

7.4.1 Peptides 7-5

7.4.2 Sample preparation: bicelles and supported lipid bilayer 7-5

7.5 Oriented solid-state NMR spectroscopy to study the conformation,topology and dynamics of peptides in membranes

7-7

7.6 Sample heterogeneity and orientational distributions 7-10

7.7 Membrane lipid composition 7-12

7.8 Taking peptide motions into consideration 7-14

7.9 Signal enhancement by DNP 7-15

Acknowledgement 7-16

References 7-16

8 Solid-state NMR and dynamic nuclear polarization studiesof molecular interactions in membranes

8-1

Vivien Yeh and Boyan B Bonev

8.1 Introduction 8-1

8.2 Membranes in the presence of small organic molecules 8-4

Solid-State NMR

viii

8.3 Membrane-active proteins and peptides 8-4

8.4 Transmembrane proteins 8-5

8.5 Membrane studies by NMR 8-6

8.6 Native membranes 8-7

8.7 Wideline and oriented membranes NMR 8-8

8.7.1 Deuterium NMR 8-8

8.7.2 Lipid phase analysis by 31P wideline NMR 8-9

8.7.3 High resolution NMR in oriented membranes – 15N PISEMA 8-11

8.8 High resolution sample spinning solid-state NMR 8-11

8.8.1 Membrane studies by 13C MAS NMR 8-12

8.8.2 Membrane interactions monitored by 31P MAS NMR 8-15

8.8.3 Correlation NMR spectroscopy in membranes 8-17

8.9 Dynamic nuclear polarization 8-21

8.10 Summary 8-23

Acknowledgements 8-23

References 8-23

9 Mapping dynamics in membrane proteins withsolid-state NMR: methods and examples

9-1

Kaustubh R Mote and P K Madhu

9.1 Introduction 9-1

9.2 NMR strategies to monitor dynamics 9-3

9.3 Monitoring dynamics with CP-based methods 9-6

9.3.1 WISE 9-6

9.3.2 CPPI 9-7

9.3.3 LG-CP 9-8

9.3.4 Dynamics in oriented NMR 9-8

9.4 Monitoring dynamics with heteronuclear DDrecoupling-based methods

9-10

9.4.1 DIPSHIFT 9-10

9.4.2 REDOR 9-12

9.4.3 Symmetry-based schemes 9-13

9.5 Monitoring dynamics with CSA recoupling methods 9-14

9.5.1 2DCSA and SUPER 9-15

9.5.2 Symmetry-based schemes for CSA recoupling 9-16

9.6 Examples of studying dynamics in membrane proteins 9-17

9.6.1 Cross-polarisation based filtering 9-17

9.6.2 Rotational-echo based experiments 9-17

Solid-State NMR

ix

9.6.3 Protein structures using SLF experiments 9-19

9.7 Schemes for higher MAS and stronger couplings 9-20

9.7.1 REDOR-schemes with dipole–dipole coupling scaling 9-21

9.7.2 Equivalence of DIPSHIIT and REDOR pulse schemes 9-22

9.7.3 DIPSHIFT and REDOR at higher MAS frequencies 9-24

9.8 Conclusions 9-25

Acknowledgements 9-25

References 9-26

10 Solid-state NMR studies of peripherally membrane-associatedproteins: dealing with dynamics, disorder and dilute conditions

10-1

Patrick C A van der Wel

10.1 Peripheral and conditional membrane proteins 10-1

10.1.1 What are peripheral membrane proteins? 10-1

10.1.2 Structural studies of peripheral membrane proteins 10-2

10.2 Solid-state NMR studies of peripheral membrane proteins 10-3

10.3 Case studies of ssNMR on peripheral membrane proteins 10-5

10.3.1 Lipid-bound cytochrome c as a pro-apoptoticlipid peroxidase

10-5

10.3.1.1 Preparing samples for MAS ssNMR 10-8

10.3.1.2 Balancing sample size, concentration andbiological relevance

10-9

10.3.1.3 Optimizing the MAS ssNMR experimentalconditions

10-9

10.3.2 Myelin basic protein 10-10

10.3.3 The Pleckstrin homology domain of PLC-δ1 10-12

10.4 Lessons learned 10-14

10.5 Conclusions 10-16

Acknowledgements 10-16

References 10-16

11 Structural dynamics of G protein-coupled receptors in lipidmembranes investigated by solid-state NMR spectroscopy

11-1

Daniel Huster

11.1 Introduction 11-2

11.2 Preparative options for studying GPCRs by NMR spectroscopic tools 11-3

11.2.1 Construct optimization and expression systems 11-3

11.2.2 Isotopic labeling 11-5

Solid-State NMR

x

11.2.3 Reconstitution into membranes and membrane mimetics 11-7

11.3 Methodological options for studying dynamics of GPCRsby solid-state NMR

11-8

11.3.1 Isotropic chemical shift changes 11-10

11.3.2 Influence of motions on anisotropic chemicalshift interactions

11-10

11.3.3 Molecular order parameters 11-11

11.3.4 Relaxation rates 11-12

11.4 Examples 11-12

11.4.1 Chemokine receptor CXCR1 11-13

11.4.2 Neuropeptide Y receptor type 2 11-14

11.4.3 Growth hormone secretagogue receptor 11-16

11.5 Conclusions 11-18

Acknowledgement 11-19

References 11-19

12 Hybridizing isotropic and anisotropic solid-stateNMR restraints for membrane protein structure determination

12-1

Daniel K Weber, Erik K Larsen, Tata Gopinath and Gianluigi Veglia

12.1 Oriented-sample solid-state NMR (OS-ssNMR) structuredetermination of membrane proteins

12-2

12.2 Chemical shift anisotropy 12-4

12.3 Dipolar coupling 12-8

12.4 Scaling of orientational restraints due to conformationaland topological dynamics

12-9

12.5 Hybridization of isotropic and anisotropic restraints 12-12

12.6 Summary 12-15

Acknowledgments 12-16

References 12-16

13 Beyond structure: understanding dynamics in membraneprotein complexes

13-1

Nikita Malik and Lynmarie Thompson

13.1 Introduction 13-1

13.2 Membrane protein dynamics 13-3

13.3 NMR studies of dynamics of specific membrane protein systems 13-7

13.3.1 Anabaena sensory rhodopsin 13-7

Solid-State NMR

xi

13.3.2 Outer membrane proteins 13-11

13.3.3 Bacterial transmembrane chemoreceptors 13-13

13.4 Conclusions 13-16

Acknowledgement 13-17

References 13-17

14 Solid-state NMR methods for investigations ofmembrane protein structure and dynamics

14-1

Peng Xiao and Vladimir Ladizhansky

14.1 Introduction 14-1

14.2 Structure determination 14-2

14.2.1 Sample preparation 14-2

14.2.2 Dipolar recoupling techniques 14-4

14.2.3 Resonance assignments 14-6

14.2.4 NMR structural restraints 14-10

14.3 Proton detection in membrane proteins 14-14

14.4 Dynamics 14-17

14.4.1 Protein dynamics 14-17

14.4.2 NMR methods for studying protein dynamics 14-19

14.4.3 Opportunity for observing mobile regions inmembrane proteins

14-22

14.5 Conclusions and perspective 14-23

Acknowledgements 14-23

References 14-24

15 Proton-detected solid-state NMR and its applications tomembrane proteins

15-1

Julia Kotschy and Rasmus Linser

15.1 Introduction 15-1

15.2 Fast MAS and deuteration 15-2

15.3 Spectroscopic possibilities of proton-detected ssNMR 15-6

15.3.1 General trends in proton-detected ssNMR 15-6

15.3.2 Signal assignment in proton-detected ssNMR 15-9

15.3.3 H/D exchange and interactions with small molecules 15-13

15.3.4 Protein structure calculation in proton-detected ssNMR 15-13

15.3.5 Relaxation and elucidation of protein dynamics 15-17

Solid-State NMR

xii

15.4 Proton detection for membrane proteins 15-19

15.4.1 General considerations 15-19

15.4.2 Preparative issues 15-20

15.4.2.1 Deuterated expression and H/D exchange 15-20

15.4.2.2 Solubilization, reconstitution and crystallizationof membrane proteins

15-21

15.4.3 Example studies using ssNMR for membrane proteins 15-23

15.4.4 Perspectives 15-27

References 15-28

Solid-State NMR

xiii

Preface

Nuclear magnetic resonance (NMR) is crucial for providing structural and dynamicinformation of biomolecules at an atomic level. Increasingly, solid-state NMR isgrowing in importance in biology as a result of new developments in technology thathave led to significant gains in sensitivity. As an essentially non-invasive spectro-scopy, solid-state NMR allows investigation of large supramolecular complexes,from cell membranes to megadalton membrane-bound proteins, at atomic resolu-tion. Solid-state NMR is fast becoming the benchmark for studies of the interplaybetween biomolecules and the role of dynamics in a biological function and cellularmechanisms. Novel approaches, techniques and analyses are being rapidly devel-oped to overcome limitations and move to another level of biological questions.

This eBook delivers a range of exciting contributions at the forefront ofbiomembrane research that push the boundaries of solid-state NMR and alsoprovide refined and detailed practical pointers for tackling complex biologicalquestions. The chapters are authored by leaders in the field and are designed tocater for both beginners and experts. The book includes studies of the orientation,conformation, dynamics and interaction of lipids, peptides and proteins in mem-brane systems; novel solid-state NMR techniques, including developments inresolution and sensitivity enhancement as well as computational tools and dynamicanalyses of biological molecules; and recent advances in high-resolution structuredetermination of membrane-associated proteins and peptides by multidimensionalsolid-state NMR spectroscopy.

The chapters cover four main areas of biological solid-state NMR: (i) applica-tions to biological membranes and whole cells, (ii) membrane active peptides,(iii) dynamics of membrane proteins, and (iv) high-resolution NMR of membraneproteins. The journey of biological solid-state NMR and its application tobiomembranes is stimulating and resonates with enthusiasm as the authors sharetheir latest discoveries and provide a roadmap for solving key biological questions.

Finally, we must thank all the authors for their magnificent efforts, cooperationand patience while the chapters were being reviewed. We also thank Professor MibelAguilar for inviting us to edit this book as part of the BPS–IOP eBooks programseries as well as the editorial team at IOP Publishing for their encouragement andassistance in putting the book together: Sarah Armstrong, Jessica Friccione andMichael Slaughter.

Marc-Antoine Sani and Frances Separovic

University of Melbourne, Australia

xiv

Foreword

Biomembranes: the Holy Grail

Life would not exist without membranes. From the most primitive prebioticenvelopes to the double membranes surrounding human nuclei, all cells or cellcompartments need to have a specific ‘skin’ that provides them with existence andfunction.

The quest for this strange skin started with the isolation of fatty acids andphospholipids two centuries ago by Chevreul and Gobley. Langmuir further showedthat lipids could form monolayers at air–water interfaces and the existence of a lipidbilayer in water was postulated by Gorter and Grendel in 1925. Intrinsic andattached proteins were added later to the model by Danielli and Davson and a‘unified’ picture was proposed by Bothorel and Belle in 1968 and by Singer andNicolson in 1972 where membrane proteins would appear rather static in a dynamicflat lipid bilayer. Nowadays, the model has been enriched by considering thedynamic nature of proteins and the fact that membranes are heterogenous in theircomposition and dynamics: specific lipids and embedded proteins may formdomains of very different dynamics, called ‘rafts’, that convey essential biologicalfunctions.

As a result, the initial two-dimensional image of a lipid–protein bilayer hasevolved. Even though this is the most abundant topology, monolayers in pulmonaryalveola have been identified and non-bilayer structures may be found in Golgi andEndoplasmic Reticulum or during contacts and fusions between cells or cellorganelles.

This rich topology landscape is not a surprise because, from a very general pointof view, these lipid–protein assemblages in water belong to the class of thermotropicand lyotropic liquid-crystals, i.e., to say their phases, in a Gibbs thermodynamicsense, can vary as a function of temperature and composition (dilution). Non-lamellar phases of hexagonal or cubic topology have been found. Under specificconditions, membrane fragments can even be isolated: they consist of flat sub-micronic bilayers capable of harboring proteins and are called bicelles or nanodiscs.

Biomembranes are thus halfway between liquids and crystals: they keep the orderof orientation but have lost their order of position because their components diffusein the plane of the membrane. This particular property is ideal for choosing nuclearmagnetic resonance (NMR) as the technique of choice to study them. In contrast tox-ray crystallography, electron microscopy and vibrational spectroscopy, NMRprovides both an atomic and overall structural description of the membranecomponents and, most importantly, a dynamic view of the movements over morethan ten time decades, from picosecond to seconds, accurately illustrating theisomerization of the bonds or the membrane fluctuations and cell fusion at themicron-to-millimeter scale. It should also be mentioned that NMR can work onwhole systems in their native state, i.e., performing NMR experiments on living cellsusing the isotope labeling strategy and examining the response of selected isotopes ina jungle of other atoms.

xv

Professor Frances Separovic and Dr Marc-Antoine Sani have gathered in thisbook many illustrations of NMR applications, primarily solid-state NMR, to studythe structure and dynamics of membranes. Approximately 35 specialists worldwidehave contributed to 15 chapters providing "l’état de l’art" of NMR applied tobiomembranes. Although NMR relaxation (T1Z, T2E, T1D, T1Q, T2CP, etc) candescribe many motional modes in membranes it was chosen not to be presented herebut to focus mainly on structure and dynamics of membrane proteins as obtainedfrom mono- or multidimensional spectra.

Different facets of solid-state NMR are presented here: they are divided betweenstatic (wide line spectroscopy) and magic angle sample spinning (MAS) experiments.The technology of spinning the rotor at the magic angle has made tremendousprogress in recent years and a proton-resolved 2D-experiement has been recentlyobtained on a membrane protein in the solid state by Pintacuda and Loquet groups.They succeeded in spinning the rotor at 111 kHz in a 1 GHz NMR machine, thusallowing one to average the static dipolar interaction and obtain proton lines of afew Hz width with increased sensitivity.

In this book wonderful examples of resolving membrane protein structure usingMAS solid-state NMR are presented by the groups of Madhu, Veglia, Thompson,Linser and Ladizhansky. On the other hand, valuable topological and dynamicinformation can be obtained from static, wide line, NMR of membranes hostingpeptides or proteins. While intrinsic orientation information is lost with MASexperiments, this information can be obtained from experiments on membranesaligned in between glass plates, oriented sample (OS) solid-state NMR, ormembranes that are magnetically oriented by the magnetic field, such as bicelles.The orientation and dynamics of protein helical segments with respect to the planeof the membrane can thus be very easily obtained. After the pioneering work of theOpella group, excellent examples are given in this book by Huster, Bechinger andVeglia, the latter nicely combining MAS and OS solid-state NMR.

As described above, this information can also be obtained from the naturallyoccurring isotopes, 1H, 31P, 13C and 14N, and allows the extraction of lipid–proteininteractions in the membrane because phospholipids are the main components.Gröbner and Williamson present a fine overview, and specific examples of the use ofNMR to decipher the mechanism of action of host defense peptides and the bindingof peripheral proteins to the membrane lipid domains are given by the Straus andvan der Wel groups.

NMR has a low intrinsic sensitivity compared to other techniques, such as massspectrometry or vibrational spectroscopy. However, the latter are not very quanti-tative, and NMR has the advantage of being isotope specific, i.e., isotope enrich-ment, which is readily available, allows monitoring a selected isotope in a forest ofother atoms. NMR is well suited to study the behavior of a single labeled molecule innatural complex systems. This labelling strategy is well known in liquid-state NMRfor the determination of the structure of soluble proteins or the identification ofmetabolic pathways. It was successfully applied 50 years ago by Seelig whopioneered deuterium NMR of lipid bilayers by showing that order parameterscould accurately describe the microdynamics of membranes. In the 1980s, the Davis

Solid-State NMR

xvi

and Dufourc groups consolidated this theory by showing that lipid phase diagramsand order parameters obtained from carbon–deuterium segments could be analyzedaccording to the de Gennes concept of order parameters in liquid crystals and couldlead to valuable information on membrane phases, bilayer thickness and membraneelasticity. In this book, the deuterium labeling strategy is illustrated by the work ofThewalt, Morrow and Booth, the latter demonstrating that deuterium isotopelabelling and solid-state NMR could be extended to whole bacterial cells.Following this labeling strategy, the Ulrich group has also applied fluorine-19NMR to biomembranes, taking advantage of the high sensitivity of this nucleus. Inthis monograph, a particular application of isotope labeling (13C, 15N) to microalgaemembranes is also described by Marcotte’s group. NMR filtering using adaptedpulse sequences allows obtaining valuable information on the structure anddynamics of membranes and identification of membrane components in thiscomplex natural system.

As already mentioned, sensitivity is an issue in solid-state NMR experiments.However, this problem is seriously beginning to be addressed by taking advantage ofDNP (dynamic nuclear polarization). The enormous polarization transfer fromelectrons of a stable organic radical (added to the membrane sample) in closeproximity to the nuclei of interest can increase the NMR signal-to-noise ratio byseveral orders on magnitude. After the pioneering works of Griffin and Oschkinat,the groups of Bechinger and Bonev describe applications to peptides withinoriented-membranes and combination of DNP and MAS NMR for low concen-trations in membranes. Following on from improvements in NMR signal forbiological systems that are difficult to obtain at high concentrations for intrinsicreasons, solid-state NMR cryoprobes are being developed by several manufacturers.The sensitivity gain is nonetheless lower than that obtained with DNP. However, itdoes not require addition of an external organic radical nor lowering the sampletemperature below biologically relevant temperatures. The first experiments are stillto be presented to the scientific community and I am sure that the two editors of thisbook will include these new perspectives in a future edition.

I wish you a good journey through these pages and I hope that you will get asclose as possible to the Holy Grail.

Erick Dufourc, Paris

26th August 2020

Solid-State NMR

xvii

Editor biographies

Frances Separovic

Professor Emeritus Frances Separovic AO FAA School ofChemistry, Bio21 InstituteUniversity of Melbourne, VIC 3010, AustraliaEmail: fs@unimelb.edu.au Phone: +61 3 9035 7539Professor Frances Separovic is a Biophysical Chemist and deputydirector of the Bio21 Institute, University of Melbourne,Australia. Her research group studies the structure and dynamics

of molecules in biological membranes, with a focus on peptide antibiotics andtoxins, primarily using solid-state NMR techniques. Frances is a Fellow of theBiophysical Society, International Society of Magnetic Resonance and theAustralian Academy of Science; an IUPAC Distinguished Women of Chemistryand an Officer of the Order of Australia. http://separovic.chemistry.unimelb.edu.au

Marc-Antoine Sani

Dr Marc-Antoine SaniSchool of Chemistry, Bio21 InstituteUniversity of Melbourne, VIC 3010, AustraliaEmail: msani@unimelb.edu.au Phone: +61 3 9034 2402Dr Marc-Antoine Sani is a senior research fellow at the School ofChemistry, University of Melbourne. His expertise precessesaround DNP NMR and solid-state NMR of biomolecules, with a

particular focus on in-cell studies. He obtained his PhD jointly from UmeåUniversity and Bordeaux University. He is an editorial board member ofANZMAGazine and the Victorian representative for the Australian Society forBiophysics.

xviii

List of contributors

Sergii AfoninKarlsruhe Institute of Technology (KIT), Institute of Biological Interfaces(IBG-2) and Institute of Organic Chemistry (IOC), P.O. Box 3644, 76021Karlsruhe, Germany

Alexandre A ArnoldDepartment of Chemistry, Université du Québec à Montréal, P.O. Box 8888,Montréal, QC, H3C 3P8 Canada

Burkhard BechingerUniversité de Strasbourg/CNRS, UMR7177, Institut de Chimie, 4, rue BlaisePascal, 67070 Strasbourg, France

Boyan B BonevSchool of Life Sciences, University of Nottingham, Queen’s Medical Centre,Nottingham NG7 2UH, UK

Valerie BoothDepartment of Biochemistry and Department of Physics and PhysicalOceanography, Memorial University of Newfoundland, St. John’s, NL, A1B3X9, Canada

Erick J DufourcInstitute of Chemistry and Biology of Membranes and Nanoobjects, CNRS,University of Bordeaux, Bordeaux Polytechnic Institute, Bordeaux, France

Tata GopinathDepartment of Biochemistry, Molecular Biology, and Biophysics, University ofMinnesota, Minneapolis, MN 55455, USA

Stephan L GrageKarlsruhe Institute of Technology (KIT), Institute of Biological Interfaces(IBG-2) and Institute of Organic Chemistry (IOC), P.O. Box 3644, 76021Karlsruhe, Germany

Gerhard GröbnerDepartment of Chemistry, Umeå University, Umeå, Sweden

Daniel HusterInstitute of Medical Physics and Biophysics, University of Leipzig, Härtelstr.16-18, D-04107 Leipzig, Germany

Julia KotschyFaculty of Chemistry and Chemical Biology, Technical University Dortmund,Otto-Hahn-Straße 4a, 44227 Dortmund, Germany

xix

Prashant KumarDepartment of Chemistry, University of British Columbia, 2036 Main Mall,Vancouver, BC, V6T 1Z1, Canada

Vladimir LadizhanskyDepartment of Physics and Biophysics Interdepartmental Group, University ofGuelph, Guelph, ON N1G 2W1 Canada

Erik K LarsenDepartment of Biochemistry, Molecular Biology, and Biophysics, University ofMinnesota, Minneapolis, MN 55455, USA

Rasmus LinserFaculty of Chemistry and Chemical Biology, Technical University Dortmund,Otto-Hahn-Straße 4a, 44227 Dortmund, Germany

P K MadhuTata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village,Ranga Reddy District, Hyderabad-500 046, India

Nikita MalikDepartment of Chemistry, University of Massachusetts Amherst, Amherst, MA01003, USA

Isabelle MarcotteDepartment of Chemistry, Université du Québec à Montréal, P.O. Box 8888,Montréal, QC, H3C 3P8 Canada

Michael R MorrowDepartment of Physics & Physical Oceanography, Memorial University ofNewfoundland, St. John’s, Newfoundland A1B 3X7, Canada

Kaustubh R MoteTata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village,Ranga Reddy District, Hyderabad-500 046, India

Alexandre PoulhazanDepartment of Chemistry, Université du Québec à Montréal, P.O. Box 8888,Montréal, QC, H3C 3P8 Canada

Evgeniy SalnikovUniversité de Strasbourg/CNRS, UMR7177, Institut de Chimie, 4, rue BlaisePascal, 67070Strasbourg, France

Suzana K StrausDepartment of Chemistry, University of British Columbia, 2036 Main Mall,Vancouver, BC, V6T 1Z1, Canada

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Allen TakayesuDepartment of Chemistry, University of British Columbia, 2036 Main Mall,Vancouver, BC, V6T 1Z1, Canada

Jennifer L ThewaltDepartment of Physics and Department of Molecular Biology & Biochemistry,Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

Lynmarie ThompsonDepartment of Chemistry, University of Massachusetts Amherst, Amherst, MA01003, USA

Anne S UlrichKarlsruhe Institute of Technology, Institute of Biological Interfaces IBG-2 andInstitute of Organic Chemistry, P.O. Box 3644, 76021 Karlsruhe, Germany

Patrick C A van der WelZernike Institute for Advanced Materials, University of Groningen, Nijenborgh4, 9747 AG Groningen, The Netherlands

Gianluigi VegliaDepartment of Biochemistry, Molecular Biology and Biophysics, Department ofChemistry, University of Minnesota, Minneapolis, MN 55455, USA

Dror E WarschawskiDepartment of Chemistry, Université du Québec à Montréal, P.O. Box 8888,Montréal, QC, H3C 3P8 CanadaSorbonne Université, École Normale Supérieure, PSL University, CNRS,Laboratoire des biomolécules, LBM, 75005 Paris, France

Daniel K WeberDepartment of Biochemistry, Molecular Biology, and Biophysics, University ofMinnesota, Minneapolis, MN 55455, USA

Philip WilliamsonCentre for Biological Sciences, University of Southampton, Southampton,United Kingdom

Peng XiaoDepartment of Physics and Biophysics Interdepartmental Group, University ofGuelph, Guelph, ON N1G 2W1 Canada

Vivien YehSchool of Life Sciences, University of Nottingham, Queen’s Medical Centre,Nottingham NG7 2UH, UK

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