Protein NMR (eBook)

Preface 5
Contents 7
Contributors 8
Assignment of Protein NMR Spectra Using Heteronuclear NMR—A Tutorial 10
1 Introduction 10
2 Scope of This Chapter 11
3 The 1H–15N HSQC—A Fingerprint of the Protein 12
4 The Triple Resonance Assignment Method 15
4.1 Optimising Parameters for Triple Resonance Experiments 16
4.2 Viewing 3D Data Sets and Generating Strip Plots and Peak Lists 18
4.3 Backbone Assignments Using 1HN, 15N, 13C? and 13CO 19
4.4 Resonance Assignments Using 1HN, 15N, 13C?, 13C?, 1H? and 1H? 23
4.5 Obtaining Further Side-Chain Assignments Using Triple Resonance Experiments 29
4.6 A Worked Example 30
4.7 Completing Side-Chain 13C/1H Assignments Using 13C HCCH–COSY/TOCSY 30
4.8 Triple Resonance Methods with Deuteration 35
4.9 Triple Resonance Methods for Proteins Above 40 kD 36
5 The Sequential Assignment Method 37
5.1 2D and 3D Experiments for 15N-Labelled Proteins 38
5.2 Stage 1: Spin System Identification from 3D TOCSY–HSQC and 3D HNHA 39
5.3 Stage 2: Sequence-Specific Assignment from 3D NOESY–HSQC and HSQC–NOESY–HSQC 41
6 Conclusion 47
References 48
Mapping Out Protein Hydration Dynamics by Overhauser Dynamic Nuclear Polarization 52
1 Introduction 52
1.1 Biophysical Tools to Study Protein Hydration 54
1.2 Introduction to ODNP 57
2 Methodology 58
2.1 Theory of ODNP in Solution 58
2.2 Experimental Quantification of ODNP in Solution 66
3 Application to Protein Folding, Protein Aggregation, Membrane Protein Topology, and Membrane–Protein Interaction 70
4 Opportunities of Overhauser DNP for Studying Protein Hydration 76
References 78
Relaxation Dispersion NMR Spectroscopy 84
1 Introduction 84
1.1 History—Development of Relaxation Dispersion NMR 85
1.2 Theoretical Aspects of Chemical Exchange 96
2 Relaxation Dispersion Experiments 101
2.1 Nitrogen-Based Relaxation Dispersion Experiments 101
2.2 Carbon and Proton-Based Relaxation Dispersion Experiments 111
2.3 Deriving the Sign of ? 118
3 Overview of the Different Methods to Probe Chemical Exchange 120
4 Applications 122
5 Perspectives 134
References 135
Solution PRE NMR 142
1 Introduction 142
2 Theory 143
3 Implementation 147
3.1 Soluble Probes 147
3.1.1 Oxygen 148
3.1.2 Aminoxyl Radicals 148
3.1.3 Metal Ions 149
3.2 NMR Measurement and Processing 151
3.2.1 Pulse Sequence Design 151
3.2.2 Processing 151
3.2.3 Signal Enhancement 152
3.2.4 Spectral Editing 154
4 Data Interpretation and Applications 154
4.1 Validation 155
4.2 Structure Calculation 157
4.3 Complexes Interfaces 158
4.4 Structure Determination in Membrane-Mimetics 159
4.5 Dynamics 160
5 Outlook 161
References 162
Line Narrowing in Oriented-Sample NMRof Membrane Proteins 167
1 Introduction 167
1.1 Why Study Membrane Proteins in Their Native-like Bilayer Environment? 167
1.2 Solid-State NMR Methods for Structure Determination of Membrane Proteins 169
1.3 Structural Information from Solid-State NMR of Macroscopically Aligned Samples 170
2 Requirements for High-Resolution Oriented-Sample NMR Spectra of Membrane Proteins 171
2.1 Effects of Uniaxial Rotational Diffusion on Solid-State NMR Spectra of Membrane Proteins 172
3 Membrane Protein Alignment Methods 178
3.1 Optimization of AAO Substrates with Homogeneous Pore Distribution for Oriented-Sample NMR 180
3.2 Round Sample Holder for Oriented-Sample NMR of Membrane Proteins Aligned by AAO Nanopores 181
3.3 Reconstitution of Lipids Bilayers and Membrane Proteins into AAO Nanopores 184
4 Lipid-Induced Conformational Changes of Pf1 Bacteriophage Coat Protein Reconstituted in Nanopore-Confined Bilayers Revealed by Solid-State NMR Spectroscopy 185
5 Conclusions and Outlook 187
References 188