Professor Gordon Roberts is Head of the School of Biological Sciences, University of Leicester. Dr Christina Redfield is Reader, Oxford Centre for Molecular Sciences, University of Oxford.

• List of Contributors xiiiIntroduction 1Lu-Yun Lian and Gordon RobertsReferences 41 Sample Preparation, Data Collection and Processing 5Frederick W. Muskett1.1 Introduction 51.2 Sample Preparation 51.2.1 Initial Considerations 61.2.2 Additives 71.2.3 Sample Conditions 71.2.4 Special Cases 81.2.5 NMR Sample Tubes 91.2.5.1 3 mm Tubes 101.3 Data Collection 111.3.1 Locking 111.3.2 Tuning 111.3.3 Shimming 121.3.4 Calibrating Pulses 131.3.5 Acquisition Parameters 141.3.6 Fast Acquisition Methods 161.4 Data Processing 17References 202 Isotope Labelling 23Mitsuhiro Takeda and Masatsune Kainosho2.1 Introduction 232.2 Production Methods for Isotopically Labelled Proteins 242.2.1 Recombinant Protein Expression in Living Organisms 242.2.1.1 Escherichia coli 242.2.1.2 Yeast Cells 252.2.1.3 Other Host Cells 252.2.2 Cell-Free Synthesis 25Protocol 1: Preparation of the Amino Acid Free S30 Extract 26Protocol 2: Cell-Free Reaction on a Small Scale 282.3 Uniform Isotope Labelling of Proteins 292.3.1 Uniform 15N Labelling 292.3.2 Uniform 13C, 15N Labelling 302.3.3 2H Labelling 302.4 Selective Isotope Labelling of Proteins 322.4.1 Amino Acid Type-Selective Labelling 322.4.2 Reverse Labelling 342.4.3 Stereo-Selective Labelling 362.5 Segmental Labelling 372.6 SAIL Methods 382.6.1 Concept of SAIL 382.6.2 Practical Procedure for the SAIL Method 41Protocol 3: Production of SAIL Proteins by the E. coliCell-Free Method 412.6.3 Residue-Selective SAIL Method 42Protocol 4: Optimisation of the Amount of SAIL Amino Acids for the Production of Calmodulin Selectively Labelled by SAIL Phenylalanine 452.7 Concluding Remarks 45Acknowledgements 46References 463 Resonance Assignments 55Lu-Yun Lian and Igor L. Barsukov3.1 Introduction 553.2 Resonance Assignment of Unlabelled Proteins 563.2.1 Spin System Assignments 573.2.2 Sequence-Specific Assignments 593.2.3 Possible Difficulties 603.3 15 N-Edited Experiments 603.4 Triple Resonance 623.4.1 3D Triple Resonance 623.4.1.1 Identification of Spin Systems 643.4.1.2 Sequential Assignment 683.4.1.3 Proline Residues 743.4.2 4D Triple Resonance 743.4.3 Computer-Assisted Backbone Assignments 763.4.4 Unstructured Proteins 763.4.5 Large Proteins 773.5 Side-Chain Assignments 77References 814 Measurement of Structural Restraints 83Geerten W. Vuister, Nico Tjandra, Yang Shen, Alex Grishaev and Stephan Grzesiek4.1 Introduction 834.2 NOE-Based Distance Restraints 844.2.1 Physical Background 844.2.2 NMR Experiments for Measuring the NOE 864.2.3 Set-up of NOESY Experiments 874.2.3.1 Estimation of T2s 87Recipe 4.1: 1–1 Echo Experiment 88Recipe 4.2: Set-up of Optimal Acquisition Times 89Recipe 4.3: Set-up of a 3D 15 N-Edited NOESY Experiment (Figure 4.2a) 90Recipe 4.4: Set-up of a 3D 13 C-Edited NOESY Experiment 914.2.4 Deriving Structural Information from NOE Cross-peaks 92Recipe 4.5: Extraction of Distances Using Classes 95Recipe 4.6: Extraction of Distances Using the Two-Spin Approximation 954.2.5 Information Content of NOE Restraints 954.3 Dihedral Restraints Derived from J-Couplings 964.3.1 Physical Background 964.3.2 NMR Experiments for Measuring J-Couplings 97Recipe 4.7: E.COSY Experiment 98Recipe 4.8: Quantitative J-Correlation 1004.3.3 Deriving Structural Information from J-Couplings 1024.4 Hydrogen Bond Restraints 1034.4.1 NMR H-Bond Observables 1034.4.2 Detection of N HO¼C H-Bonds in Proteins 104Recipe 4.9: Setting up a Long-Range HNCO Experiment for H-Bond Detection 1064.5 Orientational Restraints 1074.5.1 Physical Background 1084.5.1.1 Dipolar Couplings in Anisotropic Solution 1084.5.1.2 The Alignment Tensor 1094.5.1.3 Chemical Shifts in Anisotropic Solution 1114.5.2 Alignment Methods 1124.5.2.1 Intrinsic Molecular Alignment 1124.5.2.2 Indirect Alignment by External Media 1134.5.3 Measurements and Data Analysis 1164.5.4 Determination of the Alignment Tensor 1184.5.4.1 Degeneracy of Solutions 1214.5.4.2 Prediction of the Alignment Tensor from the Structure 1214.5.5 RDCs in Structure Validation 1224.5.5.1 Q-Factor 1224.5.5.2 Using RDC Values for Database Screening 1224.5.6 RDCs in Structure Determination 1224.5.6.1 Structure Refinement 1224.5.6.2 Domain Orientation 1254.5.6.3 De Novo Structure Determination 1284.5.7 Conclusion 1294.6 Chemical Shift Structural Restraints 1294.6.1 Origin of Chemical Shifts and Its Relation to Protein Structure 1294.6.2 Obtaining Chemical Shifts 1314.6.3 Backbone Dihedral Angle Restraints from Chemical Shifts (TALOS) 132Recipe 4.10: Using the TALOS + Program (for details see http://spin.niddk.nih.gov/bax/software/TALOS + /) 1324.6.4 Protein Structure Determination from Chemical Shifts (CS-Rosetta) 134Recipe 4.11: CS-Rosetta Structure Calculation 1364.7 Solution Scattering Restraints 1374.7.1 Physical Background 1374.7.2 Shape Reconstructions from Solution Scattering Data 1394.7.3 Use of SAXS in High-Resolution Structure Determination 1404.7.4 Sample Preparation 1414.7.5 Data Collection 1424.7.6 Data Processing and Initial Analysis 145Acknowledgement 147References 1475 Calculation of Structures from NMR Restraints 159Peter Guntert5.1 Introduction 1595.2 Historical Development 1615.3 Structure Calculation Algorithms 1645.3.1 Molecular Dynamics Simulation versus NMR Structure Calculation 1645.3.2 Potential Energy – Target Function 1655.3.3 Torsion Angle Dynamics 1665.3.3.1 Tree Structure 1675.3.3.2 Kinetic Energy 1675.3.3.3 Forces = Torques =– Gradient of the Target Function 1695.3.3.4 Equations of Motion 1695.3.3.5 Torsional Accelerations 1705.3.3.6 Time Step 1715.3.4 Simulated Annealing 172Protocol for Simulated Annealing 1725.4 Automated NOE Assignment 1735.4.1 Ambiguity of Chemical Shift Based NOESY Assignment 1745.4.2 Ambiguous Distance Restraints 1755.4.3 Combined Automated NOE Assignment and Structure Calculation with CYANA 1755.4.4 Network-Anchoring 1775.4.5 Constraint Combination 1775.4.6 Structure Calculation Cycles 1775.5 Nonclassical Approaches 1785.5.1 Assignment-Free Methods 1785.5.2 Methods Based on Residual Dipolar Couplings 1795.5.3 Chemical Shift-Based Structure Determination 1805.6 Fully Automated Structure Analysis 181References 1856 Paramagnetic Tools in Protein NMR 193Peter H.J. Keizers and Marcellus Ubbink6.1 Introduction 1936.2 Types of Restraints 1946.2.1 Paramagnetic Dipolar Relaxation Enhancement 1946.2.2 Other Types of Relaxation 1976.2.3 Residual Dipolar Couplings 1976.2.4 Contact and Pseudocontact Shifts 1996.3 What Metals to Use? 2006.4 Paramagnetic Probes 2036.4.1 Substitution of Metals 2036.4.2 Free Probes 2046.4.3 Nitroxide Labels 2046.4.4 Metal Binding Peptides 2056.4.5 Synthetic Metal Chelating Tags 206Protocol for the Application of Paramagnetic NMR on Diamagnetic Proteins 2076.5 Examples 2096.5.1 Structure Determination of Paramagnetic Proteins 2096.5.2 Structure Determination Using Artificial Paramagnets 2096.5.3 Structures of Protein Complexes 2106.5.4 Studying Dynamics with Paramagnetism 2116.6 Conclusions and Perspective 212References 2137 Structural and Dynamic Information on Ligand Binding 221Gordon Roberts7.1 Introduction 2217.2 Fundamentals of Exchange Effects on NMR Spectra 2227.2.1 Definitions 2227.2.2 Lineshape 2257.2.3 Identification of the Exchange Regime 2277.3 Measurement of Equilibrium and Rate Constants 2297.3.1 Lineshape Analysis 2297.3.1.1 Slow Exchange 2297.3.1.2 Fast Exchange 2307.3.2 Magnetisation Transfer Experiments 2317.3.2.1 Saturation Transfer 2337.3.2.2 Inversion Transfer 2337.3.2.3 Two-Dimensional Exchange Spectroscopy 2337.3.3 Relaxation Dispersion Experiments 2357.4 Detecting Binding – NMR Screening 2387.4.1 Detecting Binding by Changes in Rotational and Translational Mobility of the Ligand 2397.4.2 Detecting Binding by Magnetisation Transfer 2407.4.2.1 Saturation Transfer Difference (STD) Spectroscopy 2407.4.2.2 Water-LOGSY 2417.5 Mechanistic Information 2417.5.1 Problems of Fast Exchange 2427.5.2 Identification of Kinetic Mechanisms 2427.5.2.1 Slow Exchange 2437.5.2.2 Fast Exchange 2437.6 Structural Information 2467.6.1 Ligand Conformation – the Transferred NOE 2467.6.1.1 Exchange Rate 2487.6.1.2 Contributions from Other Species 2497.6.1.3 Spin Diffusion 2507.6.1.4 Structure Calculation 2517.6.2 Interligand Transferred NOEs 2517.6.2.1 Two Ligands Bound Simultaneously 2527.6.2.2 Competitive Ligands – INPHARMA 2527.6.3 Ligand Conformation – Transferred Cross-Correlated Relaxation 2537.6.4 Chemical Shift Mapping – Location of the Binding Site 2537.6.5 Paramagnetic Relaxation Experiments 2547.6.6 Isotope-Filtered and -Edited Experiments 256References 2598 Macromolecular Complexes 269Paul C. Driscoll8.1 Introduction 2698.2 Spectral Simplification through Differential Isotope Labelling 2708.3 Basic NMR Characterisation of Complexes 273Protocol for Protein–Protein Titrations 2738.4 3D Structure Determination of Macromolecular Protein–Ligand Complexes 2778.4.1 NOEs 2778.4.2 Saturation Transfer 2828.4.3 Residual Dipolar Couplings 2868.4.4 Paramagnetic Relaxation Enhancements 2898.4.5 Pseudo-Contact Shifts 2918.4.6 Data-Driven Docking 2938.4.7 Small Angle X-Ray Scattering (SAXS) 2968.5 Literature Examples 2978.5.1 Protein–Protein Interactions 2978.5.2 Protein–DNA Interactions 3018.5.3 Protein–RNA Interaction 3038.5.3.1 Protein–dsRNA 3038.5.3.2 Protein–ssRNA 305References 3109 Studying Partially Folded and Intrinsically Disordered Proteins Using NMR Residual Dipolar Couplings 319Malene Ringkjøbing Jensen, Valery Ozenne, Loic Salmon, Gabrielle Nodet, Phineus Markwick, Pau Bernadó and Martin Blackledge9.1 Introduction 3199.2 Ensemble Descriptions of Unfolded Proteins 3209.3 Experimental Techniques for the Characterisation of IDPs 3209.4 NMR Spectroscopy of Intrinsically Disordered Proteins 3219.4.1 Chemical Shifts 3219.4.2 Scalar Couplings 3229.4.3 Nuclear Overhauser Enhancements 3229.4.4 Paramagnetic Relaxation Enhancements 3229.4.5 Residual Dipolar Couplings 3239.5 Residual Dipolar Couplings 3239.5.1 Interpretation of RDCs in Disordered Proteins 3249.5.2 RDCs in Highly Flexible Systems: Explicit Ensemble Models 3279.5.3 RDCs to Detect Deviation from Random Coil Behaviour in IDPs 3299.5.4 Multiple RDCs Increase the Accuracy of Determination of Local Conformational Propensity 3339.5.5 Quantitative Analysis of Local Conformational Propensities from RDCs 3359.5.6 Conformational Sampling in the Disordered Transactivation Domain of p 53 3399.6 Conclusions 340References 340Index 347

"This is a must for anyone interested in using solution NMR to study proteins. Summing Up: Highly recommended. Graduate students, researchers/faculty, and professionals/practitioners." (Choice, 1 April 2012)

Professor Gordon Roberts is Head of the School of Biological Sciences, University of Leicester. Dr Christina Redfield is Reader, Oxford Centre for Molecular Sciences, University of Oxford.

• List of Contributors xiiiIntroduction 1Lu-Yun Lian and Gordon RobertsReferences 41 Sample Preparation, Data Collection and Processing 5Frederick W. Muskett1.1 Introduction 51.2 Sample Preparation 51.2.1 Initial Considerations 61.2.2 Additives 71.2.3 Sample Conditions 71.2.4 Special Cases 81.2.5 NMR Sample Tubes 91.2.5.1 3 mm Tubes 101.3 Data Collection 111.3.1 Locking 111.3.2 Tuning 111.3.3 Shimming 121.3.4 Calibrating Pulses 131.3.5 Acquisition Parameters 141.3.6 Fast Acquisition Methods 161.4 Data Processing 17References 202 Isotope Labelling 23Mitsuhiro Takeda and Masatsune Kainosho2.1 Introduction 232.2 Production Methods for Isotopically Labelled Proteins 242.2.1 Recombinant Protein Expression in Living Organisms 242.2.1.1 Escherichia coli 242.2.1.2 Yeast Cells 252.2.1.3 Other Host Cells 252.2.2 Cell-Free Synthesis 25Protocol 1: Preparation of the Amino Acid Free S30 Extract 26Protocol 2: Cell-Free Reaction on a Small Scale 282.3 Uniform Isotope Labelling of Proteins 292.3.1 Uniform 15N Labelling 292.3.2 Uniform 13C, 15N Labelling 302.3.3 2H Labelling 302.4 Selective Isotope Labelling of Proteins 322.4.1 Amino Acid Type-Selective Labelling 322.4.2 Reverse Labelling 342.4.3 Stereo-Selective Labelling 362.5 Segmental Labelling 372.6 SAIL Methods 382.6.1 Concept of SAIL 382.6.2 Practical Procedure for the SAIL Method 41Protocol 3: Production of SAIL Proteins by the E. coliCell-Free Method 412.6.3 Residue-Selective SAIL Method 42Protocol 4: Optimisation of the Amount of SAIL Amino Acids for the Production of Calmodulin Selectively Labelled by SAIL Phenylalanine 452.7 Concluding Remarks 45Acknowledgements 46References 463 Resonance Assignments 55Lu-Yun Lian and Igor L. Barsukov3.1 Introduction 553.2 Resonance Assignment of Unlabelled Proteins 563.2.1 Spin System Assignments 573.2.2 Sequence-Specific Assignments 593.2.3 Possible Difficulties 603.3 15 N-Edited Experiments 603.4 Triple Resonance 623.4.1 3D Triple Resonance 623.4.1.1 Identification of Spin Systems 643.4.1.2 Sequential Assignment 683.4.1.3 Proline Residues 743.4.2 4D Triple Resonance 743.4.3 Computer-Assisted Backbone Assignments 763.4.4 Unstructured Proteins 763.4.5 Large Proteins 773.5 Side-Chain Assignments 77References 814 Measurement of Structural Restraints 83Geerten W. Vuister, Nico Tjandra, Yang Shen, Alex Grishaev and Stephan Grzesiek4.1 Introduction 834.2 NOE-Based Distance Restraints 844.2.1 Physical Background 844.2.2 NMR Experiments for Measuring the NOE 864.2.3 Set-up of NOESY Experiments 874.2.3.1 Estimation of T2s 87Recipe 4.1: 1–1 Echo Experiment 88Recipe 4.2: Set-up of Optimal Acquisition Times 89Recipe 4.3: Set-up of a 3D 15 N-Edited NOESY Experiment (Figure 4.2a) 90Recipe 4.4: Set-up of a 3D 13 C-Edited NOESY Experiment 914.2.4 Deriving Structural Information from NOE Cross-peaks 92Recipe 4.5: Extraction of Distances Using Classes 95Recipe 4.6: Extraction of Distances Using the Two-Spin Approximation 954.2.5 Information Content of NOE Restraints 954.3 Dihedral Restraints Derived from J-Couplings 964.3.1 Physical Background 964.3.2 NMR Experiments for Measuring J-Couplings 97Recipe 4.7: E.COSY Experiment 98Recipe 4.8: Quantitative J-Correlation 1004.3.3 Deriving Structural Information from J-Couplings 1024.4 Hydrogen Bond Restraints 1034.4.1 NMR H-Bond Observables 1034.4.2 Detection of N HO¼C H-Bonds in Proteins 104Recipe 4.9: Setting up a Long-Range HNCO Experiment for H-Bond Detection 1064.5 Orientational Restraints 1074.5.1 Physical Background 1084.5.1.1 Dipolar Couplings in Anisotropic Solution 1084.5.1.2 The Alignment Tensor 1094.5.1.3 Chemical Shifts in Anisotropic Solution 1114.5.2 Alignment Methods 1124.5.2.1 Intrinsic Molecular Alignment 1124.5.2.2 Indirect Alignment by External Media 1134.5.3 Measurements and Data Analysis 1164.5.4 Determination of the Alignment Tensor 1184.5.4.1 Degeneracy of Solutions 1214.5.4.2 Prediction of the Alignment Tensor from the Structure 1214.5.5 RDCs in Structure Validation 1224.5.5.1 Q-Factor 1224.5.5.2 Using RDC Values for Database Screening 1224.5.6 RDCs in Structure Determination 1224.5.6.1 Structure Refinement 1224.5.6.2 Domain Orientation 1254.5.6.3 De Novo Structure Determination 1284.5.7 Conclusion 1294.6 Chemical Shift Structural Restraints 1294.6.1 Origin of Chemical Shifts and Its Relation to Protein Structure 1294.6.2 Obtaining Chemical Shifts 1314.6.3 Backbone Dihedral Angle Restraints from Chemical Shifts (TALOS) 132Recipe 4.10: Using the TALOS + Program (for details see http://spin.niddk.nih.gov/bax/software/TALOS + /) 1324.6.4 Protein Structure Determination from Chemical Shifts (CS-Rosetta) 134Recipe 4.11: CS-Rosetta Structure Calculation 1364.7 Solution Scattering Restraints 1374.7.1 Physical Background 1374.7.2 Shape Reconstructions from Solution Scattering Data 1394.7.3 Use of SAXS in High-Resolution Structure Determination 1404.7.4 Sample Preparation 1414.7.5 Data Collection 1424.7.6 Data Processing and Initial Analysis 145Acknowledgement 147References 1475 Calculation of Structures from NMR Restraints 159Peter Guntert5.1 Introduction 1595.2 Historical Development 1615.3 Structure Calculation Algorithms 1645.3.1 Molecular Dynamics Simulation versus NMR Structure Calculation 1645.3.2 Potential Energy – Target Function 1655.3.3 Torsion Angle Dynamics 1665.3.3.1 Tree Structure 1675.3.3.2 Kinetic Energy 1675.3.3.3 Forces = Torques =– Gradient of the Target Function 1695.3.3.4 Equations of Motion 1695.3.3.5 Torsional Accelerations 1705.3.3.6 Time Step 1715.3.4 Simulated Annealing 172Protocol for Simulated Annealing 1725.4 Automated NOE Assignment 1735.4.1 Ambiguity of Chemical Shift Based NOESY Assignment 1745.4.2 Ambiguous Distance Restraints 1755.4.3 Combined Automated NOE Assignment and Structure Calculation with CYANA 1755.4.4 Network-Anchoring 1775.4.5 Constraint Combination 1775.4.6 Structure Calculation Cycles 1775.5 Nonclassical Approaches 1785.5.1 Assignment-Free Methods 1785.5.2 Methods Based on Residual Dipolar Couplings 1795.5.3 Chemical Shift-Based Structure Determination 1805.6 Fully Automated Structure Analysis 181References 1856 Paramagnetic Tools in Protein NMR 193Peter H.J. Keizers and Marcellus Ubbink6.1 Introduction 1936.2 Types of Restraints 1946.2.1 Paramagnetic Dipolar Relaxation Enhancement 1946.2.2 Other Types of Relaxation 1976.2.3 Residual Dipolar Couplings 1976.2.4 Contact and Pseudocontact Shifts 1996.3 What Metals to Use? 2006.4 Paramagnetic Probes 2036.4.1 Substitution of Metals 2036.4.2 Free Probes 2046.4.3 Nitroxide Labels 2046.4.4 Metal Binding Peptides 2056.4.5 Synthetic Metal Chelating Tags 206Protocol for the Application of Paramagnetic NMR on Diamagnetic Proteins 2076.5 Examples 2096.5.1 Structure Determination of Paramagnetic Proteins 2096.5.2 Structure Determination Using Artificial Paramagnets 2096.5.3 Structures of Protein Complexes 2106.5.4 Studying Dynamics with Paramagnetism 2116.6 Conclusions and Perspective 212References 2137 Structural and Dynamic Information on Ligand Binding 221Gordon Roberts7.1 Introduction 2217.2 Fundamentals of Exchange Effects on NMR Spectra 2227.2.1 Definitions 2227.2.2 Lineshape 2257.2.3 Identification of the Exchange Regime 2277.3 Measurement of Equilibrium and Rate Constants 2297.3.1 Lineshape Analysis 2297.3.1.1 Slow Exchange 2297.3.1.2 Fast Exchange 2307.3.2 Magnetisation Transfer Experiments 2317.3.2.1 Saturation Transfer 2337.3.2.2 Inversion Transfer 2337.3.2.3 Two-Dimensional Exchange Spectroscopy 2337.3.3 Relaxation Dispersion Experiments 2357.4 Detecting Binding – NMR Screening 2387.4.1 Detecting Binding by Changes in Rotational and Translational Mobility of the Ligand 2397.4.2 Detecting Binding by Magnetisation Transfer 2407.4.2.1 Saturation Transfer Difference (STD) Spectroscopy 2407.4.2.2 Water-LOGSY 2417.5 Mechanistic Information 2417.5.1 Problems of Fast Exchange 2427.5.2 Identification of Kinetic Mechanisms 2427.5.2.1 Slow Exchange 2437.5.2.2 Fast Exchange 2437.6 Structural Information 2467.6.1 Ligand Conformation – the Transferred NOE 2467.6.1.1 Exchange Rate 2487.6.1.2 Contributions from Other Species 2497.6.1.3 Spin Diffusion 2507.6.1.4 Structure Calculation 2517.6.2 Interligand Transferred NOEs 2517.6.2.1 Two Ligands Bound Simultaneously 2527.6.2.2 Competitive Ligands – INPHARMA 2527.6.3 Ligand Conformation – Transferred Cross-Correlated Relaxation 2537.6.4 Chemical Shift Mapping – Location of the Binding Site 2537.6.5 Paramagnetic Relaxation Experiments 2547.6.6 Isotope-Filtered and -Edited Experiments 256References 2598 Macromolecular Complexes 269Paul C. Driscoll8.1 Introduction 2698.2 Spectral Simplification through Differential Isotope Labelling 2708.3 Basic NMR Characterisation of Complexes 273Protocol for Protein–Protein Titrations 2738.4 3D Structure Determination of Macromolecular Protein–Ligand Complexes 2778.4.1 NOEs 2778.4.2 Saturation Transfer 2828.4.3 Residual Dipolar Couplings 2868.4.4 Paramagnetic Relaxation Enhancements 2898.4.5 Pseudo-Contact Shifts 2918.4.6 Data-Driven Docking 2938.4.7 Small Angle X-Ray Scattering (SAXS) 2968.5 Literature Examples 2978.5.1 Protein–Protein Interactions 2978.5.2 Protein–DNA Interactions 3018.5.3 Protein–RNA Interaction 3038.5.3.1 Protein–dsRNA 3038.5.3.2 Protein–ssRNA 305References 3109 Studying Partially Folded and Intrinsically Disordered Proteins Using NMR Residual Dipolar Couplings 319Malene Ringkjøbing Jensen, Valery Ozenne, Loic Salmon, Gabrielle Nodet, Phineus Markwick, Pau Bernadó and Martin Blackledge9.1 Introduction 3199.2 Ensemble Descriptions of Unfolded Proteins 3209.3 Experimental Techniques for the Characterisation of IDPs 3209.4 NMR Spectroscopy of Intrinsically Disordered Proteins 3219.4.1 Chemical Shifts 3219.4.2 Scalar Couplings 3229.4.3 Nuclear Overhauser Enhancements 3229.4.4 Paramagnetic Relaxation Enhancements 3229.4.5 Residual Dipolar Couplings 3239.5 Residual Dipolar Couplings 3239.5.1 Interpretation of RDCs in Disordered Proteins 3249.5.2 RDCs in Highly Flexible Systems: Explicit Ensemble Models 3279.5.3 RDCs to Detect Deviation from Random Coil Behaviour in IDPs 3299.5.4 Multiple RDCs Increase the Accuracy of Determination of Local Conformational Propensity 3339.5.5 Quantitative Analysis of Local Conformational Propensities from RDCs 3359.5.6 Conformational Sampling in the Disordered Transactivation Domain of p 53 3399.6 Conclusions 340References 340Index 347

"This is a must for anyone interested in using solution NMR to study proteins. Summing Up: Highly recommended. Graduate students, researchers/faculty, and professionals/practitioners." (Choice, 1 April 2012)

Professor Gordon Roberts is Head of the School of Biological Sciences, University of Leicester. Dr Christina Redfield is Reader, Oxford Centre for Molecular Sciences, University of Oxford.

• List of Contributors xiiiIntroduction 1Lu-Yun Lian and Gordon RobertsReferences 41 Sample Preparation, Data Collection and Processing 5Frederick W. Muskett1.1 Introduction 51.2 Sample Preparation 51.2.1 Initial Considerations 61.2.2 Additives 71.2.3 Sample Conditions 71.2.4 Special Cases 81.2.5 NMR Sample Tubes 91.2.5.1 3 mm Tubes 101.3 Data Collection 111.3.1 Locking 111.3.2 Tuning 111.3.3 Shimming 121.3.4 Calibrating Pulses 131.3.5 Acquisition Parameters 141.3.6 Fast Acquisition Methods 161.4 Data Processing 17References 202 Isotope Labelling 23Mitsuhiro Takeda and Masatsune Kainosho2.1 Introduction 232.2 Production Methods for Isotopically Labelled Proteins 242.2.1 Recombinant Protein Expression in Living Organisms 242.2.1.1 Escherichia coli 242.2.1.2 Yeast Cells 252.2.1.3 Other Host Cells 252.2.2 Cell-Free Synthesis 25Protocol 1: Preparation of the Amino Acid Free S30 Extract 26Protocol 2: Cell-Free Reaction on a Small Scale 282.3 Uniform Isotope Labelling of Proteins 292.3.1 Uniform 15N Labelling 292.3.2 Uniform 13C, 15N Labelling 302.3.3 2H Labelling 302.4 Selective Isotope Labelling of Proteins 322.4.1 Amino Acid Type-Selective Labelling 322.4.2 Reverse Labelling 342.4.3 Stereo-Selective Labelling 362.5 Segmental Labelling 372.6 SAIL Methods 382.6.1 Concept of SAIL 382.6.2 Practical Procedure for the SAIL Method 41Protocol 3: Production of SAIL Proteins by the E. coliCell-Free Method 412.6.3 Residue-Selective SAIL Method 42Protocol 4: Optimisation of the Amount of SAIL Amino Acids for the Production of Calmodulin Selectively Labelled by SAIL Phenylalanine 452.7 Concluding Remarks 45Acknowledgements 46References 463 Resonance Assignments 55Lu-Yun Lian and Igor L. Barsukov3.1 Introduction 553.2 Resonance Assignment of Unlabelled Proteins 563.2.1 Spin System Assignments 573.2.2 Sequence-Specific Assignments 593.2.3 Possible Difficulties 603.3 15 N-Edited Experiments 603.4 Triple Resonance 623.4.1 3D Triple Resonance 623.4.1.1 Identification of Spin Systems 643.4.1.2 Sequential Assignment 683.4.1.3 Proline Residues 743.4.2 4D Triple Resonance 743.4.3 Computer-Assisted Backbone Assignments 763.4.4 Unstructured Proteins 763.4.5 Large Proteins 773.5 Side-Chain Assignments 77References 814 Measurement of Structural Restraints 83Geerten W. Vuister, Nico Tjandra, Yang Shen, Alex Grishaev and Stephan Grzesiek4.1 Introduction 834.2 NOE-Based Distance Restraints 844.2.1 Physical Background 844.2.2 NMR Experiments for Measuring the NOE 864.2.3 Set-up of NOESY Experiments 874.2.3.1 Estimation of T2s 87Recipe 4.1: 1–1 Echo Experiment 88Recipe 4.2: Set-up of Optimal Acquisition Times 89Recipe 4.3: Set-up of a 3D 15 N-Edited NOESY Experiment (Figure 4.2a) 90Recipe 4.4: Set-up of a 3D 13 C-Edited NOESY Experiment 914.2.4 Deriving Structural Information from NOE Cross-peaks 92Recipe 4.5: Extraction of Distances Using Classes 95Recipe 4.6: Extraction of Distances Using the Two-Spin Approximation 954.2.5 Information Content of NOE Restraints 954.3 Dihedral Restraints Derived from J-Couplings 964.3.1 Physical Background 964.3.2 NMR Experiments for Measuring J-Couplings 97Recipe 4.7: E.COSY Experiment 98Recipe 4.8: Quantitative J-Correlation 1004.3.3 Deriving Structural Information from J-Couplings 1024.4 Hydrogen Bond Restraints 1034.4.1 NMR H-Bond Observables 1034.4.2 Detection of N HO¼C H-Bonds in Proteins 104Recipe 4.9: Setting up a Long-Range HNCO Experiment for H-Bond Detection 1064.5 Orientational Restraints 1074.5.1 Physical Background 1084.5.1.1 Dipolar Couplings in Anisotropic Solution 1084.5.1.2 The Alignment Tensor 1094.5.1.3 Chemical Shifts in Anisotropic Solution 1114.5.2 Alignment Methods 1124.5.2.1 Intrinsic Molecular Alignment 1124.5.2.2 Indirect Alignment by External Media 1134.5.3 Measurements and Data Analysis 1164.5.4 Determination of the Alignment Tensor 1184.5.4.1 Degeneracy of Solutions 1214.5.4.2 Prediction of the Alignment Tensor from the Structure 1214.5.5 RDCs in Structure Validation 1224.5.5.1 Q-Factor 1224.5.5.2 Using RDC Values for Database Screening 1224.5.6 RDCs in Structure Determination 1224.5.6.1 Structure Refinement 1224.5.6.2 Domain Orientation 1254.5.6.3 De Novo Structure Determination 1284.5.7 Conclusion 1294.6 Chemical Shift Structural Restraints 1294.6.1 Origin of Chemical Shifts and Its Relation to Protein Structure 1294.6.2 Obtaining Chemical Shifts 1314.6.3 Backbone Dihedral Angle Restraints from Chemical Shifts (TALOS) 132Recipe 4.10: Using the TALOS + Program (for details see http://spin.niddk.nih.gov/bax/software/TALOS + /) 1324.6.4 Protein Structure Determination from Chemical Shifts (CS-Rosetta) 134Recipe 4.11: CS-Rosetta Structure Calculation 1364.7 Solution Scattering Restraints 1374.7.1 Physical Background 1374.7.2 Shape Reconstructions from Solution Scattering Data 1394.7.3 Use of SAXS in High-Resolution Structure Determination 1404.7.4 Sample Preparation 1414.7.5 Data Collection 1424.7.6 Data Processing and Initial Analysis 145Acknowledgement 147References 1475 Calculation of Structures from NMR Restraints 159Peter Guntert5.1 Introduction 1595.2 Historical Development 1615.3 Structure Calculation Algorithms 1645.3.1 Molecular Dynamics Simulation versus NMR Structure Calculation 1645.3.2 Potential Energy – Target Function 1655.3.3 Torsion Angle Dynamics 1665.3.3.1 Tree Structure 1675.3.3.2 Kinetic Energy 1675.3.3.3 Forces = Torques =– Gradient of the Target Function 1695.3.3.4 Equations of Motion 1695.3.3.5 Torsional Accelerations 1705.3.3.6 Time Step 1715.3.4 Simulated Annealing 172Protocol for Simulated Annealing 1725.4 Automated NOE Assignment 1735.4.1 Ambiguity of Chemical Shift Based NOESY Assignment 1745.4.2 Ambiguous Distance Restraints 1755.4.3 Combined Automated NOE Assignment and Structure Calculation with CYANA 1755.4.4 Network-Anchoring 1775.4.5 Constraint Combination 1775.4.6 Structure Calculation Cycles 1775.5 Nonclassical Approaches 1785.5.1 Assignment-Free Methods 1785.5.2 Methods Based on Residual Dipolar Couplings 1795.5.3 Chemical Shift-Based Structure Determination 1805.6 Fully Automated Structure Analysis 181References 1856 Paramagnetic Tools in Protein NMR 193Peter H.J. Keizers and Marcellus Ubbink6.1 Introduction 1936.2 Types of Restraints 1946.2.1 Paramagnetic Dipolar Relaxation Enhancement 1946.2.2 Other Types of Relaxation 1976.2.3 Residual Dipolar Couplings 1976.2.4 Contact and Pseudocontact Shifts 1996.3 What Metals to Use? 2006.4 Paramagnetic Probes 2036.4.1 Substitution of Metals 2036.4.2 Free Probes 2046.4.3 Nitroxide Labels 2046.4.4 Metal Binding Peptides 2056.4.5 Synthetic Metal Chelating Tags 206Protocol for the Application of Paramagnetic NMR on Diamagnetic Proteins 2076.5 Examples 2096.5.1 Structure Determination of Paramagnetic Proteins 2096.5.2 Structure Determination Using Artificial Paramagnets 2096.5.3 Structures of Protein Complexes 2106.5.4 Studying Dynamics with Paramagnetism 2116.6 Conclusions and Perspective 212References 2137 Structural and Dynamic Information on Ligand Binding 221Gordon Roberts7.1 Introduction 2217.2 Fundamentals of Exchange Effects on NMR Spectra 2227.2.1 Definitions 2227.2.2 Lineshape 2257.2.3 Identification of the Exchange Regime 2277.3 Measurement of Equilibrium and Rate Constants 2297.3.1 Lineshape Analysis 2297.3.1.1 Slow Exchange 2297.3.1.2 Fast Exchange 2307.3.2 Magnetisation Transfer Experiments 2317.3.2.1 Saturation Transfer 2337.3.2.2 Inversion Transfer 2337.3.2.3 Two-Dimensional Exchange Spectroscopy 2337.3.3 Relaxation Dispersion Experiments 2357.4 Detecting Binding – NMR Screening 2387.4.1 Detecting Binding by Changes in Rotational and Translational Mobility of the Ligand 2397.4.2 Detecting Binding by Magnetisation Transfer 2407.4.2.1 Saturation Transfer Difference (STD) Spectroscopy 2407.4.2.2 Water-LOGSY 2417.5 Mechanistic Information 2417.5.1 Problems of Fast Exchange 2427.5.2 Identification of Kinetic Mechanisms 2427.5.2.1 Slow Exchange 2437.5.2.2 Fast Exchange 2437.6 Structural Information 2467.6.1 Ligand Conformation – the Transferred NOE 2467.6.1.1 Exchange Rate 2487.6.1.2 Contributions from Other Species 2497.6.1.3 Spin Diffusion 2507.6.1.4 Structure Calculation 2517.6.2 Interligand Transferred NOEs 2517.6.2.1 Two Ligands Bound Simultaneously 2527.6.2.2 Competitive Ligands – INPHARMA 2527.6.3 Ligand Conformation – Transferred Cross-Correlated Relaxation 2537.6.4 Chemical Shift Mapping – Location of the Binding Site 2537.6.5 Paramagnetic Relaxation Experiments 2547.6.6 Isotope-Filtered and -Edited Experiments 256References 2598 Macromolecular Complexes 269Paul C. Driscoll8.1 Introduction 2698.2 Spectral Simplification through Differential Isotope Labelling 2708.3 Basic NMR Characterisation of Complexes 273Protocol for Protein–Protein Titrations 2738.4 3D Structure Determination of Macromolecular Protein–Ligand Complexes 2778.4.1 NOEs 2778.4.2 Saturation Transfer 2828.4.3 Residual Dipolar Couplings 2868.4.4 Paramagnetic Relaxation Enhancements 2898.4.5 Pseudo-Contact Shifts 2918.4.6 Data-Driven Docking 2938.4.7 Small Angle X-Ray Scattering (SAXS) 2968.5 Literature Examples 2978.5.1 Protein–Protein Interactions 2978.5.2 Protein–DNA Interactions 3018.5.3 Protein–RNA Interaction 3038.5.3.1 Protein–dsRNA 3038.5.3.2 Protein–ssRNA 305References 3109 Studying Partially Folded and Intrinsically Disordered Proteins Using NMR Residual Dipolar Couplings 319Malene Ringkjøbing Jensen, Valery Ozenne, Loic Salmon, Gabrielle Nodet, Phineus Markwick, Pau Bernadó and Martin Blackledge9.1 Introduction 3199.2 Ensemble Descriptions of Unfolded Proteins 3209.3 Experimental Techniques for the Characterisation of IDPs 3209.4 NMR Spectroscopy of Intrinsically Disordered Proteins 3219.4.1 Chemical Shifts 3219.4.2 Scalar Couplings 3229.4.3 Nuclear Overhauser Enhancements 3229.4.4 Paramagnetic Relaxation Enhancements 3229.4.5 Residual Dipolar Couplings 3239.5 Residual Dipolar Couplings 3239.5.1 Interpretation of RDCs in Disordered Proteins 3249.5.2 RDCs in Highly Flexible Systems: Explicit Ensemble Models 3279.5.3 RDCs to Detect Deviation from Random Coil Behaviour in IDPs 3299.5.4 Multiple RDCs Increase the Accuracy of Determination of Local Conformational Propensity 3339.5.5 Quantitative Analysis of Local Conformational Propensities from RDCs 3359.5.6 Conformational Sampling in the Disordered Transactivation Domain of p 53 3399.6 Conclusions 340References 340Index 347

"This is a must for anyone interested in using solution NMR to study proteins. Summing Up: Highly recommended. Graduate students, researchers/faculty, and professionals/practitioners." (Choice, 1 April 2012)