Biomolecules

Edited by Ivano Bertini, Kathleen S. McGreevy, and Giacomo Parigi

Towards Mechanistic Systems Biology

NMR of

57268File AttachmentCover.jpg

Edited byIvano Bertini,Kathleen S. McGreevy,and Giacomo Parigi

NMR of Biomolecules

Related Titles

Keeler, J.

Understanding NMR Spectroscopy

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de Graaf, R.

In Vivo NMR Spectroscopy

Principles and Techniques

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ISBN: 978-0-470-02670-0

Edited byIvano Bertini,Kathleen S. McGreevy,and Giacomo Parigi

NMR of Biomolecules

Towards Mechanistic Systems Biology

The Editors

Prof. Dr. Ivano BertiniUniversity of FlorenceDepartment of Chemistry andMagnetic Resonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Kathleen S. McGreevyUniversity of FlorenceDepartment of Chemistry andMagnetic Resonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Prof. Giacomo ParigiUniversity of FlorenceDepartment of Chemistry andMagnetic Resonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

CoverThe artwork on the cover attempts to convey the ideaof the systems of interacting biomolecules that are atthe basis of Life. The Birth of Venus, which has beenreimagined for the cover in this spirit, was painted bySandro Botticelli in the late 15th century and is heldby the Uffizi Gallery in Florence. His painting depictsthe birth of the goddess of love as she emerges as afully grown adult from the sea.

Acording to Plato, as well as members of theFlorentine Platonic Academy, Venus had two aspects:an earthly goddess who aroused humans to physicallove, and a heavenly goddess that inspired intellectuallove. Who better than she to represent our passionfor the study of biomolecular structures andmechanisms, and the physical-intellectual duality thatleads us to learn more about the living world aroundand within each of us? Actually Venus has alreadybeen used as the logo by the Society of BiologicalInorganic Chemistry for the Journal of BiologicalInorganic Chemistry (JBIC).

Limit of Liability/Disclaimer of Warranty: While thepublisher and author have used their best efforts inpreparing this book, they make no representations orwarranties with respect to the accuracy or complete-ness of the contents of this book and specificallydisclaim any implied warranties of merchantability orfitness for a particular purpose. No warranty can becreated or extended by sales representatives or writtensales materials. The Advice and strategies containedherein may not be suitable for your situation. Youshould consult with a professional where appropriate.Neither the publisher nor authors shall be liable forany loss of profit or any other commercial damages,including but not limited to special, incidental, con-sequential, or other damages.

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j V

Contents

Preface XXI

List of Contributors XXIII

List of Abbreviations XXIX

Part One Introduction 1

1 NMR and its Place in Mechanistic Systems Biology 3Ivano Bertini, Kathleen S. McGreevy, and Giacomo Parigi

2 Structure of Biomolecules: Fundamentals 7

2.1 Structural Features of Proteins 7Lucia Banci and Francesca Cantini

2.1.1 Introduction: From Primary to Quaternary Structure 72.1.2 Geometrical and Conformational Properties 82.1.2.1 Backbone Dihedral Angles 82.1.2.2 Side-Chain Dihedral Angles 92.1.3 Secondary Structure Elements in Proteins 92.1.4 Prediction of Secondary Structure 132.1.5 Structural Motifs and Structural Domains – Combination of Secondary Structural Elements and Structural Motifs 132.1.6 Types of Folds and their Classification 152.1.6.1 Folds of the a Class 152.1.6.2 Folds in the b Class 162.1.6.3 Folds in the a/b Class 172.1.6.4 Folds in the a þ b Class 172.1.7 Tertiary Structure 182.1.8 Quaternary Structure 19

2.2 Nucleic Acids 21Mirko Cevec, Hendrik R.A. Jonker, Senada Nozinovic, Christian Richter, and Harald Schwalbe

2.2.1 Introduction 212.2.1.1 Conformations 222.2.2 DNA Structure 242.2.2.1 B-DNA and Derivatives 242.2.2.2 A-DNA 252.2.2.3 Z-DNA 252.2.2.4 Nonstandard DNA Structures 252.2.2.4.1 Circular DNA 252.2.2.4.2 Helical Junction 262.2.2.4.3 Triple Helix 262.2.2.4.4 i-Motif 26

2.2.2.4.5 Quadruplex DNA 262.2.3 RNA Structure 272.2.3.1 Regular RNA Structure – A-Form Helices 282.2.3.2 Mismatches, Bulges, and Unusual Base Pairing 292.2.3.3 Reversal and Alteration of Strand Direction: Commonly

Observed Loop and Turn Motifs 292.2.3.3.1 U-Turn 292.2.3.3.2 K-Turn 292.2.3.3.3 C-Loop 292.2.3.3.4 E-Loop 302.2.3.4 Tetraloops and Tetraloop–Receptor Contact 302.2.3.5 Higher-Order RNA Tertiary Structure Elements:

Coaxial Stacking Motifs 312.2.3.6 DNA–RNA Hybrids 31

3 What Can be Learned About the Structure and Dynamicsof Biomolecules from NMR 33

3.1 Proteins Studied by NMR 33Lucio Ferella, Antonio Rosato, and Paola Turano

3.1.1 Why NMR Structures? 333.1.2 NMR Bundle 373.1.3 Protein Dynamics 413.1.4 Intermolecular Interactions Involving Proteins 44

3.2 Nucleic Acids Studied by NMR 47Janez Plavec

3.2.1 Structure, Mobility, and Function 47

Part Two Role of NMR in the Study of the Structure and Dynamicsof Biomolecules 51

4 Determination of Protein Structure and Dynamics 53Lucio Ferella, Antonio Rosato, and Paola Turano

4.1 Determination of Protein Structures 534.1.1 Resonance Assignment 534.2 NMR Restraints 584.2.1 Distance Restraints 584.2.2 Dihedral Angles 594.2.3 Residual Dipolar Couplings 614.3 Structure Calculations 654.3.1 Traditional 654.3.2 Automated NOESY Assignment 694.3.3 Energy Refinement of Protein Structures 704.3.4 ChemicalShift-BasedApproaches forProteinStructureDetermination 714.4 Validation of Protein Structures 724.4.1 Experimental Data 724.4.2 Geometric Quality 744.5 Protein Dynamics and NMR Observables 764.5.1 NMR Observables Affected by Dynamics 764.5.2 NMR Experiments to Measure Dynamics and their Interpretation 784.6 Protocols 834.6.1 Sample Labeling 834.6.2 NMR Assignment 834.6.3 Manual Collection of Restraints 864.6.4 Structure Calculations 874.6.5 Structure Refinement 89

VI j Contents

4.6.6 Chemical Shift-Based Structure Calculations 904.6.7 Structure Validation 904.6.8 Protein Dynamics 914.7 Troubleshooting 924.7.1 Data Collection 924.7.2 Structure Calculations 93

Further Reading 94

5 DNA 97Janez Plavec

5.1 NMR Spectroscopy of DNA 975.2 Assessment of the Folding Topology 995.3 Resonance Assignment through Sequential and Interstrand

Interactions 1005.4 Pseudorotation of Deoxyribofuranose Rings 1045.5 Backbone Conformation 1055.6 Natural Abundance Nucleobase Substitutions 1065.7 Natural Abundance Heteronuclear Experiments 1065.8 Site-Specific Low Isotopic Enrichment 1075.9 Translational Diffusion Coefficients 1075.10 Determination of Three-Dimensional Structure 1075.11 Search for Transient Structures 1095.12 Protocols 1105.12.1 Sample Preparation and Initial NMR Experiments 1105.12.2 Stoichiometric Analysis through Translational Diffusion 1115.12.3 Sequential Assignment 1115.12.4 Assessment of the Preferred Sugar Pucker 1125.12.5 Conformations along the Backbone 1125.12.6 Nucleobase Substitutions and Site-Specific Low 13C/15N

Isotopic Enrichment 1125.12.7 Topology and Atomic-Detail Three-Dimensional Structure

Determination 1135.13 Example Experiments and Troubleshooting 114

Further Reading 115

6 RNA 119Richard �Stefl and Vladim�ır Sklen�a�r

6.1 NMR Spectroscopy of RNA 1196.2 Preparation of RNA Samples for NMR 1206.2.1 In Vitro Transcription Using T7 RNA Polymerase 1206.2.2 In Vivo Recombinant RNA Synthesis 1206.2.3 Chemical Synthesis 1206.2.4 Segmental Labeling 1216.3 Probing of the RNA Fold 1216.4 Assessment of the Spectral Resolution 1226.5 Strategy for the Resonance Assignment 1236.5.1 Hydrogen Bond Formation and Base Pair Identification 1236.5.2 Through-Bond-Type Experiments – Base Spin System Identification 1246.5.3 Through-Bond-Type Experiments – Sugar Spin System Identification 1246.5.4 Sequential Connectivities 1256.6 Collection of Structural Information 1266.6.1 Collection of Distance-Dependent Structural Restraints 1266.6.2 Collection of Torsion-Angle-Dependent Structural Restraints 1276.6.3 Collection of Long-Range Structural Restraints 1276.7 Structural Calculation of RNA 1286.8 Assessment of Quality of NMR Structures 1296.9 Protocols 129

Contents j VII

6.9.1 Flowcharts 1306.9.1.1 Sample Preparation 1306.9.1.2 Collection of the Experimental Data 1306.9.1.3 Structure Calculation 1316.9.2 Protocol for In Vitro Transcription Using Bacteriophage T7 RNA

Polymerase 1326.9.2.1 Template Design 1326.9.2.2 Transcription Protocol 1336.9.2.3 Purification of RNA 1346.9.2.4 Simple Protocol for Bacteriophage T7 RNA Polymerase Preparation 1346.10 Troubleshooting 135

Further Reading 135

7 Intrinsically Disordered Proteins 137Isabella C. Felli, Roberta Pierattelli, and Peter Tompa

7.1 Intrinsically Disordered Proteins 1377.1.1 When the Concept was First Introduced 1377.1.2 Functions and Functional Advantages Associated with Disorder 1387.2 Importance of NMR to Study IDPs 1407.3 Structural and Dynamic Information on IDPs – NMR Observables 1417.3.1 Reduced Chemical Shift Dispersion and Sequence-

Specific Assignment 1417.3.2 Chemical Shifts and Secondary Structural Propensities 1427.3.3 Additional Observables and Conformational Averaging 1437.3.4 Scalar Couplings 1437.3.5 Residual Dipolar Couplings 1437.3.6 Solvent Exposure 1447.3.7 15N Relaxation and Heteronuclear NOEs 1447.3.8 Paramagnetic Relaxation Rate Enhancements 1457.3.9 Proton–Proton NOEs 1457.3.10 RelevanceofStructuralDisorder in InVivo and In-Cell Studies of IDPs 1467.4 Protocols 1467.4.1 Use of Bioinformatic Tools and Databases 1467.4.2 Use of NMR in the Characterization of IDPs 1487.5 Troubleshooting 1507.5.1 Bioinformatics Can Help! 1507.5.2 Understanding the Function of Unfolded Protein Regions 1517.5.3 Does In Vitro Reflect In Vivo Behavior? 152

Further Reading 152

8 Paramagnetic Molecules 155Ivano Bertini, Claudio Luchinat, and Giacomo Parigi

8.1 Paramagnetism-Assisted NMR 1558.2 Scalar and Dipolar Electron Spin–Nuclear Spin Interactions:

Hyperfine Shift 1578.2.1 Contact Contributions to the Hyperfine Shift 1578.2.2 Pseudocontact Contributions to the Hyperfine Shift 1588.3 Scalar and Dipolar Electron Spin–Nuclear Spin Interactions: PRE 1598.4 Indirect Electron Spin–Nuclear Spin Effects:

Paramagnetism-Induced RDCs 1618.5 Cross-Correlation Between Curie and Dipolar Relaxation 1628.6 Good Metal Ions and Bad Metal Ions 1638.7 Paramagnetism-Based Drug Discovery 1648.8 Protocols 1658.8.1 Collecting the Paramagnetism-Based Restraints 1658.8.2 Protocols to Extract Structural Information from the PCS,

PRDC, PRE and PCCR 166

VIII j Contents

8.8.3 Protocols for Protein–Protein Interactions 1678.8.4 Protocols for the Analysis of Conformational Freedom

in Two-Domain Proteins 1688.8.5 Example Experiment 1698.9 Troubleshooting 1698.9.1 Tips andTricks toOptimizeSignalDetection inParamagneticSystems 1698.9.2 Selection of the Paramagnetic Ion 1708.9.3 Switching Between Diamagnetic and Paramagnetic Systems 170

Further Reading 170

Part Three Role of NMR in the Study of the Structure and Dynamicsof Biomolecular Interactions 173

9 NMR Methodologies for the Analysis of Protein–Protein Interactions 175Tobias Madl and Michael Sattler

9.1 Introduction 1759.2 Dynamics and Ligand Binding 1769.3 General Strategy 1779.4 Overview of Methods 1789.4.1 Sample Preparation 1789.4.2 Structures of Domains/Subunits 1799.4.3 Interfaces 1809.4.3.1 Chemical Shift Perturbations (CSPs) 1809.4.3.2 NOEs 1809.4.3.3 Cross-Saturation 1819.4.3.4 Differential Line-Broadening 1819.4.3.5 Hydrogen Exchange 1829.4.3.6 Solvent PREs 1829.4.4 Domain/Subunit Orientation 1839.4.4.1 NMR Relaxation Data 1839.4.4.2 Residual Dipolar Couplings (RDCs) 1849.4.4.3 Paramagnetic Restraints 1849.4.5 Structure Calculations 1859.5 Outlook 1869.6 Protocols for the Analysis of Protein Complexes 1869.6.1 Spin Labeling and Paramagnetic Tagging 1869.6.2 Structures of the Individual Domains/Subunits 1889.6.3 Optimizing Conditions for Structural Studies of the Protein Complex 1899.6.4 Detection of Dynamics 1899.6.5 Determining Interaction Interfaces Using Solvent PREs 1899.6.6 Structure Calculation Approach 1919.6.7 Example Experiment 1939.7 Troubleshooting 194

Further Reading 194

10 Metal-Mediated Interactions 197Simone Ciofi-Baffoni

10.1 Theoretical Background 19710.2 Protocol for the Structural Determination of a

Metal-Mediated Complex 20010.2.1 Optimization of Experimental Conditions to Obtain the

Protein–Protein Complex 20010.2.2 Titrations to Map Protein–Protein Interfaces and Obtain

Binding Characteristics 20110.2.3 Modeling 20110.2.4 Definition of Metal Coordination 20110.2.5 Determination of Three-Dimensional Structure 202

Contents j IX

10.3 Example Experiment 20210.4 Troubleshooting 202

Further Reading 203

11 Protein–Paramagnetic Protein Interactions 205Peter H.J. Keizers, Yoshitaka Hiruma, and Marcellus Ubbink

11.1 Paramagnetic Sources in Protein Complexes 20511.2 Types of NMR Restraints Obtained from Paramagnetic Centers 20611.3 Protein Complexes 20711.3.1 Structures of Protein Complexes 20711.3.2 Dynamics in Protein Complexes 20811.3.2.1 Plastocyanin and Cytochrome f 20911.3.2.2 Cytochrome c and Cytochrome c Peroxidase 20911.3.2.3 Histidine Phosphocarrier Protein and Enzyme I 20911.3.2.4 Adrenodoxin and Cytochrome c 21011.3.2.5 Calmodulin Domain Dynamics 21111.4 Protocols 21111.4.1 Protein Titrations to Obtain the Kd and a Binding Map 21111.4.2 Paramagnetic Tagging and Use of Paramagnets in NMR 21211.4.3 Ensemble Modeling 21411.5 Example Experiment 21511.6 Troubleshooting 215

Further Reading 217

12 Protein–RNA Interactions 219Vijayalaxmi Manoharan, Jose Manuel P�erez-Ca~nadillas, and Andres Ramos

12.1 Introduction 21912.1.1 Post-Transcriptional Regulation and RNA Recognition

Domains 21912.1.2 Protein–RNA Interfaces 21912.2 NMR Methodology 22112.2.1 Using NMR to Investigate Protein–RNA Interactions 22112.2.2 Mapping Interaction Sites 22212.2.2.1 Chemical Shift Perturbation 22212.2.2.2 Cross-Saturation 22312.2.2.3 Paramagnetic Relaxation Enhancement 22412.2.3 Affinity and Specificity 22612.2.3.1 Determining Dissociation Constants 22612.2.3.2 Determining Stoichiometry 22612.2.3.3 Scaffold-Independent Analysis 22712.2.4 High-Resolution Structure Determination and Dynamics 22712.3 Protocols and Troubleshooting 22812.3.1 Preparation of Protein–RNA Complexes 22812.3.1.1 Materials and Sample Preparation 22912.3.1.2 Troubleshooting 22912.3.2 Protocol and Example Experiment 1: Calculating the Affinity of a

Protein–RNA Interaction 23012.3.2.1 Materials and Sample Preparation 23012.3.2.2 Troubleshooting 23112.3.3 Protocol and Example Experiment 2: Paramagnetic Labeling 23212.3.3.1 Materials and Sample Preparation 23312.3.3.2 Troubleshooting 23312.3.4 Protocol and Example Experiment 3: SIA 23412.3.4.1 Materials and Sample Preparation 23412.3.4.2 Troubleshooting 235

Further Reading 235

X j Contents

13 Protein–DNA Interactions 239Lidija Kova�ci�c and Rolf Boelens

13.1 State of the Art 23913.2 Conclusions and Perspectives 24213.3 Protocols 24313.3.1 Sample Preparation 24313.3.2 NMR Methodology 24413.3.3 Identification of the Interaction Surfaces 24713.3.4 Structure Calculations 25013.4 Troubleshooting 251

Further Reading 252

Part Four NMR in Drug Discovery 253

14 High-Throughput Screening and Fragment-Based Design:General Considerations for Lead Discovery and Optimization 255Maurizio Pellecchia

14.1 High-Throughput Screening and Fragment-Based Design 25514.2 General Aspects of NMR Spectroscopy in Hit Identification and

Optimization Processes 25714.3 Chemical Shift Perturbation as a Screening Method 261

Further Reading 263

15 Ligand-Observed NMR in Fragment-Based Approaches 265Pawe /l �Sled�z, Chris Abell, and Alessio Ciulli

15.1 Ligand-Observed NMR Spectroscopy 26515.2 On the Transient Binding of Small Molecules to the Protein 26615.3 Questions Asked by Ligand-Based Fragment Screening 26715.3.1 Direct Yes/No Binding Experiments 26815.3.1.1 STD 26915.3.1.2 WaterLOGSY 27015.3.1.3 Relaxation-Edited One-Dimensional Experiments 27115.3.2 Affinity Measurements and Affinity-Oriented Screening 27115.3.3 Information on the Binding Mode 27215.4 Summary 27415.5 Protocols 27415.5.1 General Aspects of Sample Preparation 27415.5.2 Preparing Samples 27515.5.3 Pulse Sequences 27615.5.4 Automation 27615.6 Example Experiments 27615.6.1 One-Dimensional Direct Binding Experiments 27615.6.2 Competition NMR Screening Experiments 27715.6.3 Two-Dimensional ILOE Binding Experiments 27815.7 Troubleshooting 27815.7.1 Sample Preparation 27815.7.2 Experimental Setup 280

Further Readings 280

16 Interactions of Metallodrugs with DNA 283Hong-Ke Liu and Peter J. Sadler

16.1 Metallodrugs and DNA Interactions 28316.1.1 Metallodrugs 28316.1.2 General Features of Metallodrug–DNA Interactions 28416.1.3 NMR Techniques and Metallodrug–DNA Interactions 28516.2 Coordinative Binding 28616.3 Groove Binding 289

Contents j XI

16.3.1 Major Groove Binding 28916.3.2 Minor Groove Binding 28916.4 Intercalation and Insertion 29016.4.1 Metallo-Intercalators 29016.4.2 DNA Insertion and Metallo-Inserters 29116.5 Dual Binding (Coordination and Intercalation) 29116.5.1 Intercalation by Metallodrug with s-Bonded Intercalator 29116.5.2 Intercalation by Metallodrug with p-Bonded Intercalator 29216.6 Protocols 29316.6.1 Determination of the Coordinative Binding Sites 29316.6.2 Detection of Intercalation and Insertion 29416.6.3 Detection of Metallodrug–DNA Groove-Binding

Interactions 29416.6.4 Example Experiment 29416.7 Tricks and Troubleshooting 294

Further Reading 295

17 RNA as a Drug Target 299Jan-Peter Ferner, Elke Duchardt Ferner, J€org Rinnenthal,Janina Buck, Jens W€ohnert, and Harald Schwalbe

17.1 RNA as a Target for Small Molecules 29917.2 Chemical Shift Perturbation and Paramagnetic Relaxation

Enhancement 30117.3 Nuclear Overhauser Effect-Based Methods 30417.4 Fluorine Labeling of RNA 30417.5 Ligand-Based Methods 30517.6 Protocols 30617.6.1 Determination of the Cooperativity Between Mg2þ -

Binding Sites in RNA by CSP Analysis 30617.6.2 Ligand-Binding Site Mapping by CSP Tracking 30817.6.3 Mapping Mg2þ -Binding Sites in RNA Using MnCl2

PRE Experiments 30817.6.4 Ligand-Binding Site Mapping by NOE Methods 30817.6.5 Mapping Outer Sphere Mg2þ -Binding Sites by Co

(NH3)3þ

6 NOESY Experiments 31117.7 Troubleshooting 312

Further Reading 313

18 Fluorine NMR Spectroscopy for Biochemical Screening inDrug Discovery 315Claudio Dalvit

18.1 Enzymatic Inhibition Mechanisms 31618.2 n-FABS 31718.2.1 One Substrate and One Enzyme 31818.2.2 One Substrate and One Enzyme in the Presence of Serum Albumin and/

or Enzymes of the Cytochrome P450 Superfamily 31818.2.3 One Substrate with Multiple Enzymes 31918.2.4 Multiple Substrates with One Enzyme 32018.2.5 Multiple Substrates with Multiple Enzymes 32018.2.6 Application to Functional Genomics 32118.3 Comparison of n-FABS with Other Biophysical Techniques 32218.4 Outlook 32318.5 Protocols 32318.5.1 Protocol for Setup of the n-FABS Assay 32318.5.2 Protocol for a Screening Run with n-FABS 32418.5.3 Protocol for Measuring the IC50 of Identified Inhibitors 32518.5.4 Example Experiment 326

XII j Contents

18.6 Troubleshooting 326Further Reading 327

19 NMR of Peptides 329Johannes G. Beck, Andreas O. Frank, and Horst Kessler

19.1 Introduction 32919.2 Resonance Assignment 33019.3 Stereostructure and Conformational Restraints 33019.3.1 NOEs and ROEs 33119.3.2 3J Coupling Constants 33219.3.3 Residual Dipolar Couplings 33219.4 Structure Calculation 33319.5 Importance of Peptide Conformations for Biological Activity 33419.6 Protocols 33419.6.1 Resonance Assignment 33419.6.2 ROESY: Extraction of Accurate Distances 33619.6.3 3J Couplings: Use in Structure Determination and Useful Experiments 33719.6.4 Modeling of the Structure 33919.6.4.1 Distance Geometry 34019.6.4.2 MD 34019.7 Troubleshooting 342

Further Reading 343

Part Five Solid-State NMR 345

20 Biomolecular Solid-State NMR/Basics 347Emeline Barbet-Massin and Guido Pintacuda

20.1 Introduction 34720.2 NMR Hamiltonian 34720.3 Magic Angle Spinning 34920.4 Cross-Polarization 35020.5 Heteronuclear 1H Decoupling 35020.6 Dipolar Recoupling 35120.7 Recent Progress: New Probes – Ultrafast MAS – High Magnetic Fields 35420.8 Protocols 35520.8.1 Hardware Setup 35520.8.2 Magic Angle Setting 35620.8.3 Adamantane 35620.8.3.1 MAS Spinning 35620.8.3.2 Calibrate the 1H Channel 35720.8.4 Calibrate the 13C Channel 35720.8.4.1 Reference the Spectra 35820.8.5 Cross-Polarization 35820.8.5.1 Set the Spinning 35820.8.5.2 Calibrate the 1H Channel 35820.8.5.3 Setup a Cross-Polarization Experiment 35820.8.5.4 Calibrate the 13C/15N Fields 36020.8.6 Heteronuclear Decoupling Sequences 36020.8.7 DCP 36020.8.8 Recoupling Sequences 36120.8.9 Example of an Experiment 36220.9 Troubleshooting 36220.9.1 Tips to Optimize Signal Acquisition and Sensitivity 36220.9.2 Narrowing the 13C or 15N Linewidths 36320.9.3 Recoupling Optimization 363

Further Reading 364

Contents j XIII

21 Protein Dynamics in the Solid State 367J�ozef R. Lewandowski and Lyndon Emsley

21.1 Introduction 36721.2 Basic Concepts 36921.3 Coherent versus Incoherent Processes: Decay is

not Always Relaxation 37021.4 Deuterium as a Probe of Dynamics 37121.5 15N and 13C T1 – Spin-Lattice Relaxation 37221.6 Protocols 37321.6.1 Measuring 15N T1 Relaxation 37321.6.2 Measuring 13C T1 Relaxation 37321.6.3 Heteronuclear NOE 37321.6.4 Measuring 15N Dipolar–CSA Cross-Correlated Relaxation 37421.6.5 Measuring 15N R1r 37421.6.6 Measuring Motionally Averaged Secular Interactions 37421.6.7 Slow Conformational Exchange 37421.6.8 Identification of Highly Mobile Sites 374

Further Reading 375

22 Microcrystalline Proteins – An Ideal Benchmark forMethodology Development 377W. Trent Franks, Barth-Jan van Rossum, Benjamin Bardiaux, Enrico Ravera,Giacomo Parigi, Claudio Luchinat, and Hartmut Oschkinat

22.1 Microcrystalline Protein Sample Preparation 37722.2 Sequential Assignment of Proteins 37822.3 Structural Restraints 38022.3.1 Distance Measurements: Cross-Relaxation-Like Transfer 38022.3.1.1 PDSD/DARR/RAD 38022.3.1.2 ChhC and NhhC Experiments 38122.3.1.3 1H-Detected NOE 38122.3.2 Distance Measurements: Multiple-Pulse Dipolar Recoupling 38222.3.2.1 RFDR 38222.3.2.2 TSAR, PAR, and PAIN 38222.3.2.3 REDOR 38222.3.2.4 TEDOR 38222.3.2.5 Proton Dipolar Recoupling: TMREV and LG-CP 38322.3.3 Parameters Encoding Torsion Angles 38322.3.3.1 Chemical Shifts 38322.3.3.2 Double Dip-Shift Spectroscopy 38322.3.3.3 CSA–Dipolar Interactions 38422.4 Paramagnetic Systems 38422.4.1 PRE 38422.4.2 PCS 38622.5 Benchmarking of the Solid-State NMR Structure Determination

Methodology: Comparison of Structure Calculation Protocolsand Accuracy of Structures 386

22.6 Protocols 38922.6.1 Three-Dimensional Nanocrystalline Sample Preparation 38922.6.2 Sequential Assignments of Solid-State NMR Spectra 38922.6.3 Distance Restraint Assignments with Solid-State NMR 38922.6.4 Secondary Structure Prediction by TALOS 39022.6.5 Dipolar Tensor, CSA, and Vector Angle Fitting 39022.6.6 Use of PCS for Structural Restraints: Structure of the

Catalytic Domain of MMP-12 as an Example 39022.7 Troubleshooting 391

Further Reading 392

XIV j Contents

23 Structural Studies of Protein Fibrils by Solid-State NMR 395Anja B€ockmann and Beat H. Meier

23.1 Background 39523.2 NMR Spectra of Fibrils 39623.3 Outlook 39723.4 Protocols and Examples 39823.4.1 Sample Preparation 39823.4.2 Experiments for the Resonance Assignment 39823.4.3 Experiments to Obtain Structural Restraints 40023.4.4 Flexible Elements 40123.4.5 Structure Calculation in Fibrils 40223.5 Troubleshooting 404

Further Reading 405

24 Solid-State NMR on Membrane Proteins: Methods and Applications 407A.A. Cukkemane, M. Renault, and M. Baldus

24.1 Solid-State NMR of Membrane Proteins 40724.1.1 Magic Angle Spinning 40724.1.2 Methods for High-Resolution Structural Investigation of Membrane Proteins 40824.1.3 Investigation of Membrane Protein Topology 40924.1.4 Sensitivity and Resolution 41024.2 MAS Applied to Ion Channels and Retinal Proteins 41124.2.1 Retinal Proteins 41124.2.2 Chimeric Potassium Channel KcsA–Kv1.3 41124.3 Protocols 41324.3.1 Sample Preparation 41324.3.2 Isotope Labeling 41424.3.3 Resonance Assignment 41524.3.4 Collecting NMR Restraints 41524.3.5 A Real Experiment 41624.4 Troubleshooting 41724.4.1 Tips and Tricks in Optimizing Sample Preparation 41724.4.2 Improving Sensitivity 417

Further Reading 417

Part Six Frontiers in NMR Spectroscopy 419

25 Dynamic Nuclear Polarization 421Thomas F. Prisner

25.1 Dynamic Nuclear Polarization at High Magnetic Fields 42125.2 Theoretical Background 42225.2.1 Overhauser Effect 42325.2.2 Solid Effect 42525.2.3 Three-Spin Cross-Polarization: Cross-Effect 42525.2.4 Many-Spin Cross-Polarization: Thermal Mixing 42625.3 Protocols 42725.3.1 HF Liquid DNP Spectrometers 42725.3.2 Shuttle DNP 42825.3.3 SS MAS DNP 42825.3.4 Low-Temperature Dissolution Polarizer 42925.4 Example Experiment 42925.5 Perspectives 43025.5.1 HF Liquid DNP 43025.5.2 Shuttle DNP 43025.5.3 SS MAS DNP 43025.5.4 Dissolution DNP 431

Further Reading 431

Contents j XV

26 13C Direct Detection NMR 433Isabella C. Felli and Roberta Pierattelli

26.1 13C Direct Detection NMR for Biomolecular Applications 43326.1.1 13C NMR Properties and Application Areas 43326.1.2 Problem of Homonuclear Decoupling in the Direct Acquisition

Dimension 43626.1.3 Experiments for High-Resolution NMR 43826.2 Protocols for Experimental Setup 43926.3 Troubleshooting 442

Further Reading 442

27 Speeding Up Multidimensional NMR Data Acquisition 445Bernhard Brutscher, Dominique Marion, and Lucio Frydman

27.1 Multidimensional NMR: Basic Concepts and Features 44527.1.1 Discrete Data Sampling, Aliasing, and Truncation Artifacts 44627.1.2 Time Requirements in N-Dimensional NMR 44727.2 Fast Methods in N-Dimensional NMR 44727.2.1 Sparse Time-Domain Data Sampling and Processing 44727.2.1.1 Sampling Scheme and the Point Spread Function 44827.2.1.2 Sparse Sampling Schemes 44827.2.1.3 Alternative Data Processing Methods 44927.2.2 Fast-Pulsing Methods 45127.2.2.1 Repetition Rate and Experimental Sensitivity 45227.2.2.2 Longitudinal 1H Relaxation Enhancement 45227.2.2.3 Ernst Angle Excitation 45327.2.2.4 BEST and SOFAST Experiments 45327.2.3 Single-Scan Ultrafast Two-Dimensional NMR 45427.2.3.1 General Principles 45427.2.3.2 Spatiotemporal Encoding Process 45527.2.3.3 Spatiotemporal Decoding: Two-Dimensional NMR in One Scan 45527.3 Protocols for Fast N-Dimensional NMR and Troubleshooting 45727.3.1 Setting Up an Appropriate Sampling Grid 45727.3.1.1 Information-Driven Spectral Aliasing 45827.3.1.2 Random Sparse Sampling and MaxEnt Processing 45827.3.2 Practical Considerations for the Setup of Fast-Pulsing

Experiments 45927.3.3 Setting Up a Single-Scan Two-Dimensional Experiment 46027.3.3.1 Properties of Different Encoding Schemes 46027.3.3.2 Single-Scan Two-Dimensional NMR Spectrum: Characteristics 46127.3.4 Fast Two-Dimensional NMR for

Sample Quality Screening and Molecular Interaction Studies 46227.3.5 Real-Time Two-Dimensional NMR Measurements 463

Further Reading 465

28 Metabolomics 467Leonardo Tenori

28.1 Metabolomics in Systems Biology 46728.2 NMR and Metabolomics 46928.3 Data Analysis 47128.4 Success in the Application of Metabolomics 47228.5 Protocols 47328.5.1 Serum Extraction 47328.5.2 Plasma Extraction 47328.5.3 Urine Collection 47428.5.4 Tip on Samples Labeling 47428.5.5 Sample Preparation for NMR Analysis 47428.5.5.1 Urine 474

XVI j Contents

28.5.5.2 Serum/Plasma 47528.5.6 Buffer Recipes 47528.5.7 NMR Analysis (Working with a 600-MHz Bruker Spectrometer) 47528.5.7.1 Urine 47528.5.7.2 Serum/Plasma 47528.5.8 Spectral Processing 47628.5.9 Bucketing 47628.5.10 Data Mining 47628.5.11 Example Experiment 47728.6 Troubleshooting 477

Further Reading 477

29 In-Cell Protein NMR Spectroscopy 479David S. Burz, David Cowburn, Kaushik Dutta, and Alexander Shekhtman

29.1 Background 47929.2 Specific Applications 48029.2.1 Structure Determination 48129.2.2 Perturbations within the Cell – How In-Cell and In Vitro are

Different Environments 48129.2.3 Specific Protein–Protein Interactions: STINT-NMR 48329.2.4 Other Protein–Ligand Interactions 48429.2.5 Post-Translational Modification – In-Cell Biochemistry 48429.2.5.1 Other In-Cell Biochemical Studies 48529.2.6 Nucleic Acids In-Cell 48529.3 Conclusions and Future Directions 48529.4 Protocols and Example Experiments 48629.4.1 Experimental Design of Multiple Expression Systems In-Cell 48629.4.2 Detailed STINT Protocol 48729.4.2.1 Data Acquisition and Analysis 48829.4.2.2 Example Experiment 48929.4.3 Detailed SMILI-NMR Protocol 48929.4.3.1 Example Experiment 48929.4.4 Detailed Protocol for Post-Translational Modification 49129.4.4.1 Example Experiment 49129.5 Troubleshooting 49229.5.1 No Signal 49229.5.2 Weak Signal 49329.5.3 Signal is Extracellular 49329.5.3.1 Control Experiments for Protein Leakage/Cell Lysis 49329.5.3.2 Cell Viability 49329.5.4 Cell Growth 493

Further Reading 493

30 Structural Investigation of Cell-Free Expressed Membrane Proteins 497SolmazSobhanifar, SinaReckel, FrankL€ohr, FrankBernhard, andVolkerD€otsch

30.1 Introduction 49730.2 Cell-Free Expression of Membrane Proteins 49830.3 Cell-Free Expression in Membrane-Mimetic Environments 49930.4 Strategies for Functional Protein Expression 50030.5 Cell-Free Approaches for Structural Studies 50130.6 Cell-Free Labeling Strategies for Backbone Assignment 50230.7 Structure Determination with Limited Nuclear Overhauser Effect

Long-Distance Restraints 50230.8 Protocols 50430.8.1 Extract 50430.8.2 Reaction Devices 50530.8.3 DNA Template Preparation 506

Contents j XVII

30.8.4 Different Modes for the Cell-Free Production ofMembrane Proteins 50630.8.5 Example Experiment 50730.9 Troubleshooting 507

Further Reading 508

Part Seven Computational Aspects 509

31 Grid Computing 511Antonio Rosato

31.1 Grid Infrastructure 51131.2 e-NMR Web Platform 51231.3 Protocols 51531.3.1 How to Register with the e-NMR/WeNMR VO 51531.3.2 Performing a CYANA Calculation 51631.3.3 Refining a Structure with AMBER 51631.4 Troubleshooting 517

Further Reading 518

32 Protein–Protein Docking with HADDOCK 521Christophe Schmitz, Adrien S.J. Melquiond, Sjoerd J. de Vries, Ezgi Karaca,Marc van Dijk, Panagiotis L. Kastritis, and Alexandre M.J.J. Bonvin

32.1 Protein–Protein Docking: General Concepts 52132.1.1 Why Protein–Protein Docking? 52132.1.2 General Methods for Protein–Protein Docking 52232.2 Gathering Experimental Information for Data-Driven Docking 52232.2.1 Chemical Shift Perturbations 52332.2.2 Cross-Saturation Experiments 52432.2.3 Hydrogen/Deuterium Exchange 52432.2.4 Intermolecular NOEs 52432.2.5 Paramagnetic Relaxation Enhancement 52432.2.6 Pseudocontact Shift 52532.2.7 Residual Dipolar Coupling 52532.2.8 Diffusion Anisotropy 52632.2.9 Non-NMR Information 52632.3 How Does HADDOCK Use the Information? 52632.3.1 Incorporation of Ambiguous Distance Restraints 52732.3.2 Incorporation of Unambiguous Distance Restraints 52732.3.3 Incorporation of Shape Restraints 52832.3.4 Incorporation of Orientation Restraints 52832.3.5 Symmetry Restraints 52832.3.6 Additional Docking Mode 52832.3.7 Overview of a HADDOCK Run 52932.4 Protocol: A Guided Tour of the HADDOCK Web Interface 53032.4.1 Prerequisite: Registration 53032.4.2 Description of the Web Interface 53132.4.3 Analysis of the Docking Run 53332.5 Troubleshooting 53432.5.1 General Considerations 53432.5.2 Problems Related to the PDB File 53432.5.3 Problems Encountered During Docking 535

Further Reading 535

33 Automated Protein Structure Determination Methods 537Paul Guerry and Torsten Herrmann

33.1 NMR Experiment-Driven Protein Modeling 53733.2 NOE-Based Structure Determination 53833.2.1 Chemical Shift Ambiguity 539

XVIII j Contents

33.2.2 Ambiguous Distance Restraints 53933.2.3 Using Intermediate Structures and Elimination of Artifacts 54033.2.4 Structure Calculation and Energy Refinement 54033.2.5 Structure Validation 54133.3 Sequence-Specific Resonance Assignment 54133.4 NMR Signal Identification 54233.5 Perspectives 54233.6 Protocols 54333.7 Example Structure Determination and Troubleshooting 54433.7.1 Sequence-Specific Resonance Assignment 54533.7.2 Amino Acid Sequence, Cis/Trans Isomerization, and Redox State 54533.7.3 NOE Assignment, Structure Calculation, and Structure Validation 545

Further Reading 546

34 NMR Structure Determination of Protein–Ligand Complexes 549Ulrich Schieborr, Sridhar Sreeramulu, and Harald Schwalbe

34.1 Protein–Ligand Complex Structure Determination by NMR 54934.2 Methods for High-Affinity Binders 55034.3 Methods for Low-Affinity Binders 55234.4 Protocols and Troubleshooting 558

Further Reading 561

35 Small Angle X-Ray Scattering/Small Angle Neutron Scattering as Methods Complementary to NMR 563M.V. Petoukhov and D.I. Svergun

35.1 Introduction 56335.2 Invariants 56635.3 Ab Initio Shape Determination 56735.4 Validation of Atomic Models 56835.5 Rigid-Body Modeling of Quaternary Structure 56835.6 Equilibrium Mixtures and Flexible Systems 56935.7 Protocols 57035.7.1 Validation of High-Resolution Models by Solution Scattering 57035.7.2 Unrestrained Rigid-Body Modeling of a Complex 57135.7.3 Rigid-Body Modeling with Contact Restraints from NMR Chemical Shifts 57235.7.4 Rigid-Body Modeling of a Complex with Orientational Constraints from NMR RDCs 57235.7.5 Characterization of Flexible Systems 57335.8 Troubleshooting 573

Further Reading 574

References 575

Index 609

Contents j XIX

Preface

The use of NMR to solve protein structures has a tradition that dates back to 1984(M.P. Williamson, T.F. Havel, and K. W€uthrich (1985) J. Mol. Biol. 182, 295). Sincethat time, the role of NMR in structural biology has constantly increased in terms ofthe number of researchers involved and the scientific relevance of the results.Spectrometers are becoming more and more powerful, with magnetic fields thatcurrently reach 22 T, and high-temperature superconducting materials raise thepossibility that this value can be surpassed. The investment required for a magnet ofthe above intensity is currently around D10 million (US$14.3 million) and anestimate ofD18million (US$25.7million) is reasonable for new-generationmagnets.It is clear that NMR is a technology that deserves a special place in researchinfrastructures, as individual schools may find it difficult to have a battery ofmachines, all at the forefront of the technology, dedicated to various types ofexperiments. In 1994, the EuropeanCommission (EC) began financing transnationalaccess to NMR instrumentation at some research infrastructures, which havecontinued and grown in number until the present with the EC-funded Bio-NMR1)

project. In Europe, the recent European Strategy Forum for Research Infrastructures(ESFRI) Roadmap identifiesNMR as a fundamental node in the Integrated StructuralBiology Infrastructure (INSTRUCT),2) while it also plays a role in the EU-OPEN-SCREEN (European Infrastructure of Open Screening Platforms for ChemicalBiology)3) infrastructure, Euro-BioImaging,4) and Biobanking and BiomolecularResources Research Infrastructure (BBMRI).5)

The EC-funded electronic infrastructures (e-NMR6) and WeNMR7)) providenonspecialists with tools for automatic data handling, structure calculations, molec-ular dynamics simulations, and the creation of interaction models in such a way thatthe potential of the NMR technology can blossom in favor of the progress of science.

Much of this reasoningwas debated during the FP6-fundedCoordinationActionNMR-Life8) and resulted in a booklet entitled NMR in Mechanistic Systems Biology,9)

which ultimately served as the spark for this volume. We were pleased when Gregor

1) http://www.bio-nmr.net.

2) http://www.structuralbiology.eu.

3) http://www.eu-openscreen.eu.

4) http://www.eurobioimaging.eu.

5) http://www.bbmri.eu.

6) http://www.enmr.eu.

7) http://www.wenmr.eu.

8) http://www.postgenomicnmr.net.

9) http://www.postgenomicnmr.net/NMRLife/docs/NMR_in_MSB.pdf.

j XXI

Cicchetti from Wiley-VCH noticed this booklet and proposed that we edit a book onthe very same subject, andwe gathered a number of outstanding contributors to fulfillthe task.

Our intention, which we hope pervades the book, was to provide a text forgraduate students, junior post-docs, and other newcomers that would serve as anintroduction to the field, addressing classical NMR approaches from solution to thesolid state, providing some tips and tricks not available in journal articles, andproviding perspectives on future developments. It is our hope that the Protocols andTroubleshooting sections will be of assistance and guidance when choosing experi-ments and overcoming difficulties.

However, everyone who has experience in editing books knows how difficult atask it is – obtaining the manuscripts on time, convincing everyone to adhere to atemplate and write for students and not for their fellow professors, and even drawingthe line on what content to include and when to call an end to the editorial process,including substitution of recalcitrant contributors. We editors have tried our best toovercome these difficulties, but we are aware that much more could have been done.For example, the development of isotopic labeling has been fundamental for thedevelopment of NMR, but we decided not to address it here. The reader shouldtherefore be aware that the field of NMR is even broader and more exciting than itappears from our efforts!

Part I of the book (Introduction) explains NMRs role in Mechanistic SystemsBiology and provides a broad overview of biomolecular structure before identifyingwhat NMR can teach us about the structure and dynamics of biomolecules. Parts II–VII address a series of relevant topics in NMR-driven biological research: the role ofNMR in the study of the structure and dynamics of biomolecules, its role in the studyof the structure and dynamics of biomolecular interactions, NMR in drug discovery,solid-state NMR, frontiers in NMR spectroscopy, and computational aspects.

We would like to take the opportunity to thank, in addition to Gregor, Dr. MarcoFragai of the Center forMagnetic Resonance (CERM) at theUniversity of Florence forhis assistance in editing some chapters of the book, and Professor Claudio Luchinat,who consistently demonstrates his friendship and his willingness to support anyinitiative of CERM, and the scientific personnel of CERM who have contributed todiscussions and sustained the work.

It is our sincere hope that this book will find a home not only in NMR facilities,but also in biomedical laboratories around the world, where it can be of use to thebroader scientific community and help diffuse NMR as a technique for the study ofbiological systems.

Ivano BertiniKathleen S. McGreevy

Florence, January 2012 Giacomo Parigi

XXII j Preface

List of Contributors

Chris AbellUniversity of CambridgeUniversity Chemical LaboratoryLensfield RoadCambridge CB2 1EWUK

Marc BaldusUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Lucia BanciUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Emeline Barbet-MassinUniversité de LyonInstitut de Sciences AnalitiquesCentre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Benjamin BardiauxLeibniz-Institut für MolekularePharmakologieNMR-Supported Structural BiologyRobert-Rössle-Strasse 1013125 BerlinGermany

Johannes G. BeckTechnische Universität MünchenDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Frank BernhardGoethe University FrankfurtInstitute of Biophysical Chemistry andCenter for Biomolecular MagneticResonance (BMRZ)Max-von-Laue-Strasse 960438 Frankfurt am MainGermany

Ivano BertiniUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Anja BöckmannUniversité de LyonIBCP UMR 5086 CNRS7 passage du Vercors69367 LyonFrance

Rolf BoelensUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Alexandre M.J.J. BonvinUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Bernhard BrutscherInstitut de Biologie Structurale – Jean-Pierre EbelUMR5075 CNRS-CEA-UJF41 rue Jules Horowitz38027 Grenoble CedexFrance

Janina BuckGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

David S. BurzUniversity at AlbanyDepartment of Chemistry1400 Washington AvenueAlbany, NY 12222USA

Francesca CantiniUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

j XXIII

Mirko CevecGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 FrankfurtGermany

Simone Ciofi-BaffoniUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Alessio CiulliUniversity of CambridgeUniversity Chemical LaboratoryLensfield RoadCambridge CB2 1EWUK

David CowburnYeshiva UniversityAlbert Einstein College of Medicine1300 Morris Park AvenueBronx, NY 10461USA

Abhishek A. CukkemaneUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Claudio DalvitUniversity of NeuchâtelDepartment of ChemistryAvenue de Bellevaux 512000 NeuchâtelSwitzerland

Marc van DijkUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Volker DötschGoethe University FrankfurtInstitute of Biophysical Chemistry andCenter for Biomolecular MagneticResonance (BMRZ)Max-von-Laue-Strasse 960438 Frankfurt am MainGermany

Elke Duchardt-FernerGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofMolecular BiosciencesMax-von-Laue-Straße 960438 Frankfurt am MainGermany

Kaushik DuttaNew York Structural Biology CenterNew York, NY 10027USA

Lyndon EmsleyUniversité de LyonCNRS/ENS Lyon/UCB-Lyon 1Centre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Isabella C. FelliUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Lucio FerellaUniversity of FlorenceMagnetic Resonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Jan-Peter FernerGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

Andreas O. FrankVanderbilt University School of MedicineDepartment of Biochemistry802 Robinson Research BuildingNashville, TN 37232-0146USA

W. Trent FranksLeibniz-Institut für MolekularePharmakologieNMR-Supported Structural BiologyRobert-Rössle-Strasse 1013125 BerlinGermany

Lucio FrydmanWeizmann Institute of ScienceDepartment of Chemical PhysicsChemical Science BuildingRehovot 76100Israel

Paul GuerryUniversité de LyonCNRS/ENS Lyon/UCB-Lyon 1Centre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Torsten HerrmannUniversité de LyonCNRS/ENS Lyon/UCB-Lyon 1Centre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Yoshitaka HirumaLeiden UniversityLeiden Institute of ChemistryGorlaeus LaboratoriesEinsteinweg 552333 CC LeidenThe Netherlands

Hendrik R.A. JonkerGoethe University FrankfurtJohann Wolfgang Goethe-UniversityCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 FrankfurtGermany

XXIV j List of Contributors

Ezgi KaracaUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Panagiotis L. KastritisUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Peter H.J. KeizersLeiden UniversityLeiden Institute of ChemistryGorlaeus LaboratoriesEinsteinweg 552333 CC LeidenThe Netherlands

Horst KesslerTechnische Universität MünchenDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Lidija Kova�ci�cUniversity of UtrechtBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

and

Jožef Stefan InstituteDepartment of Molecular and BiomedicalSciencesJamova cesta 391000 LjubljanaSlovenia

Józef R. LewandowskiUniversité de LyonCNRS/ENS Lyon/UCB-Lyon 1Centre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Hong-Ke LiuNanjing Normal UniversityCollege of Chemistry and MaterialsScienceJiangsu Key Laboratory of BiofunctionalMaterialsWenyuan Road 1, Nanjing 210046China

Frank LöhrGoethe University FrankfurtInstitute of Biophysical Chemistry andCenter for Biomolecular MagneticResonance (BMRZ)Max-von-Laue-Strasse 960438 Frankfurt am MainGermany

Claudio LuchinatUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Tobias MadlHelmholtz Zentrum MünchenInstitute of Structural BiologyIngolstädter Landstrasse 185764 NeuherbergGermany

and

Technische Universität MünchenBiomolecular NMR and Munich Centerfor Integrated Protein ScienceDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Vijayalaxmi ManoharanMedical Research Council NationalInstitute for Medical ResearchMolecular Structure DivisionThe RidgewayMill HillLondon NW7 1AAUK

Dominique MarionInstitut de Biologie Structurale – Jean-Pierre EbelUMR5075 CNRS-CEA-UJF41 rue Jules Horowitz38027 Grenoble CedexFrance

Kathleen S. McGreevyUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Beat H. MeierETH ZurichLaboratory of Physical ChemistryWolfgang-Pauli-Strasse 108093 ZurichSwitzerland

Adrien S.J. MelquiondUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Senada NozinovicGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 FrankfurtGermany

Hartmut OschkinatLeibniz-Institut für MolekularePharmakologieNMR-Supported Structural BiologyRobert-Rössle-Strasse 1013125 BerlinGermany

Giacomo ParigiUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Maurizio PellecchiaSanford-Burnham Medical ResearchInstitute10901 North Torrey Pines RoadLa Jolla, CA 92037USA

List of Contributors j XXV

Jose Manuel Perez-CanadillasConsejo Superior de InvestigacionesCientíficas (CSIC)Instituto de Quimica Fisica “Rocasolano”Serrano 11928006 MadridSpain

Maxim V. PetoukhovEuropean Molecular Biology LaboratoryHamburg OutstationNotkestrasse 8522607 HamburgGermany

Roberta PierattelliUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Guido PintacudaUniversité de LyonInstitut de Sciences AnalitiquesCentre de RMN à Très Hauts Champs5 rue de la Doua69100 VilleurbanneFrance

Janez PlavecNational Institute of ChemistrySlovenian NMR CenterHajdrihova 191000 LjubljanaSlovenia

and

EN-FIST Center of ExcellenceDunajska 1561000 LjubljanaSlovenia

and

University of LjubljanaFaculty of Chemistry and ChemicalTechnologyAskerceva cesta 51000 LjubljanaSlovenia

Thomas F. PrisnerGoethe University FrankfurtInstitute of Physical and TheoreticalChemistry and Center for BiomolecularMagnetic Resonance (BMRZ)Max-von-Laue-Strasse 760438 FrankfurtGermany

Andres RamosMedical Research Council NationalInstitute for Medical ResearchMolecular Structure DivisionThe RidgewayMill HillLondon NW7 1AAUK

Enrico RaveraUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Sina ReckelGoethe University FrankfurtInstitute of Biophysical Chemistry andCenter for Biomolecular MagneticResonance (BMRZ)Max-von-Laue-Strasse 960438 Frankfurt am MainGermany

Marie RenaultUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

Christian RichterJohann Wolfgang Goethe-UniversityCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 FrankfurtGermany

Jörg RinnenthalGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

Antonio RosatoUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Barth-Jan van RossumLeibniz-Institut für MolekularePharmakologieNMR-Supported Structural BiologyRobert-Rössle-Strasse 1013125 BerlinGermany

Peter J. SadlerUniversity of WarwickDepartment of ChemistryGibbet Hill RoadCoventry CV4 7ALUK

Michael SattlerHelmholtz Zentrum MünchenInstitute of Structural BiologyIngolstädter Landstrasse 185764 NeuherbergGermany

and

Technische Universität MünchenBiomolecular NMR and Munich Centerfor Integrated Protein ScienceDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Ulrich SchieborrGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

XXVI j List of Contributors

Christophe SchmitzUtrecht UniversityBijvoet Center for Biomolecular ResearchScience FacultyPadualaan 83584 CH UtrechtThe Netherlands

Harald SchwalbeGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

Alexander ShekhtmanUniversity at AlbanyDepartment of Chemistry1400 Washington AvenueAlbany, NY 12222USA

Vladimír Sklená�rMasaryk UniversityNational Center for BiomolecularResearchFaculty of Science and Central EuropeanInstitute of Technology (CEITEC)Kamenice 5625 00 BrnoCzech Republic

Paweł Śled�zUniversity of CambridgeUniversity Chemical LaboratoryLensfield RoadCambridge CB2 1EWUK

Solmaz SobhanifarGoethe University FrankfurtInstitute of Biophysical Chemistry andCenter for Biomolecular MagneticResonance (BMRZ)Max-von-Laue-Strasse 960438 Frankfurt am MainGermany

Sridhar SreeramuluGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofOrganic Chemistry and Chemical BiologyMax-von-Laue-Straße 760438 Frankfurt am MainGermany

Richard ŠteflMasaryk UniversityNational Center for BiomolecularResearchFaculty of Science and Central EuropeanInstitute of Technology (CEITEC)Kamenice 5625 00 BrnoCzech Republic

Dimitri I. SvergunEuropean Molecular Biology LaboratoryHamburg OutstationNotkestrasse 8522607 HamburgGermany

Leonardo TenoriUniversity of FlorenceMagnetic Resonance Center (CERM) andFiorGen FoundationVia L. Sacconi 650019 Sesto FiorentinoItaly

Peter TompaVIB Department of Structural BiologyVrije Universiteit BrusselPleinlaan 21050 BrusselsBelgium

and

Hungarian Academy of SciencesInstitute of EnzymologyBiological Research CenterKarolina �ut 291113 BudapestHungary

Paola TuranoUniversity of FlorenceDepartment of Chemistry and MagneticResonance Center (CERM)Via L. Sacconi 650019 Sesto FiorentinoItaly

Marcellus UbbinkLeiden UniversityLeiden Institute of ChemistryGorlaeus LaboratoriesEinsteinweg 552333 CC LeidenThe Netherlands

Sjoerd J. de VriesUtrecht UniversityBijvoet Center for Biomolecular ResearchPadualaan 83584 CH UtrechtThe Netherlands

and

Technische Universität MünchenPhysik-Department T38James Franck Straße 185748 GarchingGermany

Jens WöhnertGoethe University FrankfurtCenter for Biomolecular MagneticResonance (BMRZ) and Institute ofMolecular BiosciencesMax-von-Laue-Straße 960438 Frankfurt am MainGermany

List of Contributors j XXVII