Protein Nmr Spectroscopy - Practical Techniques And Applications

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique in structural biology for obtaining high resolution 3-D structures of proteins, second only, and complementary to X-ray crystallography. Molecules are studied in solution, where conditions are closer to what is found in the cell. It is the primary technique used to obtain information on intrinsically disordered (unfolded) proteins, since these proteins will not crystallize easily.

The aim of this book is to provide the newcomer to NMR techniques with practical guidance on how to choose the right experiment, how to carry out the experiment, and how to analyse the resulting spectra. Those who are familiar with the chemical applications of NMR will also find it helpful in describing the special requirements of proteins.

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

Introduction
Lu-Yun Lian & Gordon Roberts

1 Sample Preparation, Data Collection and Processing
Frederick W. Muskett

1.1. Introduction
1.2. Sample Preparation
1.3. Data Collection
1.4. Data Processing
References

2 Isotope Labelling
Mitsuhiro Takeda and Masatsune Kainosho

2,1. Introduction
2.2 Production Methods for Isotopically Labelled Proteins
2.3 Protocol 1: Preparation of the amino acid free S30 extract
2.4 Protocol 2: Cell-Free Reaction on a Small Scale
2.5 Uniform Isotope Labelling of Proteins
2.6 Selective Isotope Labelling of Proteins
2.7 Segmental Labelling
2.8 SAIL Methods
2.9 Protocol 3: Production of SAIL Proteins by the E. coli Cell-Free Method
2.10 Protocol. 4: Optimisation of the Amount of SAIL Amino Acids for the Production of Calmodulin Selectively Labelled by SAIL Phenylalanine
2.11 Concluding Remarks
References

3 Resonance Assignments
Lu-Yun Lian and Igor L. Barsukov

3.1 Introduction
3.2 Resonance Assignment of Unlabelled Proteins
3.3 15N-Edited Experiments
3.4 Triple Resonance
3.5. Side-Chain Assignments
References

4 Measurement of Structural Restraints
Geerten Vuister, Nico Tjandra, Yang Shen, Alex Grishaev, and Stephan Grzesiek

4.1 Introduction
4.2 NOE-Based Distance Restraints
4.3 Dihedral Restraints Derived from J-Couplings
4.4. Hydrogen Bond Restraints
4.5 Orientational Restraints
4.6 Chemical Shift Structural Restraints
4.7 Solution Scattering Restraints
References

5 Calculation of Structures from NMR Restraints
Peter Guntert

5.1. Introduction
5.2. Historical Development
5.3. Structure Calculation Algorithms
5.4. Automated NOE Assignment
5.5. Nonclassical Approaches
5.6. Fully Automated Structure Analysis
References

6 Paramagnetic Tools in Protein NMR
Peter H.J. Keizers and Marcellus Ubbink

6.1 Introduction
6.2. Types of Restraints
6.3. What Metals to Use?
6.4. Paramagnetic Probes
6.5. Protocol for the Application of Paramagnetic NMR on Diamagnetic Proteins
6.6. Examples
6.7. Conclusions and Perspective
References

7 Structural and Dynamic Information on Ligand Binding
Gordon C.K. Roberts

7.1. Introduction
7.2. Fundamentals of Exchange Effects on NMR Spectra
7.3. Measurement of Equilibrium and Rate Constants
7.4. Detecting Binding - NMR Screening
7.5. Mechanistic Information
7.6. Structural Information
References

8 Macromolecular Complexes
Paul C. Driscoll

8.1 Introduction
8.2. Spectral Simplification through Differential Isotope Labelling
8.3. Basic NMR Characterisation of Complexes
8.4. Protocol of Protein-Protein Titrations
8.5. 3D Structure Determination of Macromolecular Protein-Ligand Complexes
8.6. Literature Examples
References

9 Studying Partially Folded and Intrinsically Disordered Proteins using NMR Residual Dipolar Couplings
Malene Ringkjøbing Jensen, Valéry Ozenne, Loic Salmon, Gabrielle Nodet, Phineus Markwick, Pau Bernadó and Martin Blackledge

9.1. Introduction
9.2. Ensemble Descriptions of Unfolded Proteins
9.3. Experimental Techniques for the Characterisation of IDPs
9.4 NMR Spectroscopy of Intrinsically Disordered Proteins
9.5 Residual Dipolar Couplings
9.6. Conclusions
References