Protein NMR Spectroscopy -

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Editeur : John Wiley & Sons

Langue : Anglais

Parution : 01/08/2011

Format : Moyen, de 350g à 1kg

Nombre de pages : 368

Expédition : 816

Dimensions : 25.3 x 17.9 x 2.4

ISBN : 0470721936

Résumé :

Nuclear Magnetic Resonance (NMR) spectroscopy, a physical phenomenon based upon the magnetic properties of certain atomic nuclei, has found a wide range of applications in life sciences over recent decades. This up-to-date volume covers NMR techniques and their application to proteins, with a focus on practical details. Providing newcomers to NMR with practical guidance to carry out successful experiments with proteins and analyze the resulting spectra, those familiar with the chemical applications of NMR will also find it useful in understanding the special requirements of protein NMR.

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Biographie:

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....

Sommaire:

List of Contributors xiii

Introduction 1
Lu-Yun Lian and Gordon Roberts

References 4

1 Sample Preparation, Data Collection and Processing 5
Frederick W. Muskett

1.1 Introduction 5

1.2 Sample Preparation 5

1.2.1 Initial Considerations 6

1.2.2 Additives 7

1.2.3 Sample Conditions 7

1.2.4 Special Cases 8

1.2.5 NMR Sample Tubes 9

1.2.5.1 3 mm Tubes 10

1.3 Data Collection 11

1.3.1 Locking 11

1.3.2 Tuning 11

1.3.3 Shimming 12

1.3.4 Calibrating Pulses 13

1.3.5 Acquisition Parameters 14

1.3.6 Fast Acquisition Methods 16

1.4 Data Processing 17

References 20

2 Isotope Labelling 23
Mitsuhiro Takeda and Masatsune Kainosho

2.1 Introduction 23

2.2 Production Methods for Isotopically Labelled Proteins 24

2.2.1 Recombinant Protein Expression in Living Organisms 24

2.2.1.1 Escherichia coli 24

2.2.1.2 Yeast Cells 25

2.2.1.3 Other Host Cells 25

2.2.2 Cell-Free Synthesis 25

Protocol 1: Preparation of the Amino Acid Free S30 Extract 26

Protocol 2: Cell-Free Reaction on a Small Scale 28

2.3 Uniform Isotope Labelling of Proteins 29

2.3.1 Uniform ¹⁵N Labelling 29

2.3.2 Uniform ¹³C, ¹⁵N Labelling 30

2.3.3 ²H Labelling 30

2.4 Selective Isotope Labelling of Proteins 32

2.4.1 Amino Acid Type-Selective Labelling 32

2.4.2 Reverse Labelling 34

2.4.3 Stereo-Selective Labelling 36

2.5 Segmental Labelling 37

2.6 SAIL Methods 38

2.6.1 Concept of SAIL 38

2.6.2 Practical Procedure for the SAIL Method 41

Protocol 3: Production of SAIL Proteins by the E. coli

Cell-Free Method 41

2.6.3 Residue-Selective SAIL Method 42

Protocol 4: Optimisation of the Amount of SAIL Amino Acids for the Production of Calmodulin Selectively Labelled by SAIL Phenylalanine 45

2.7 Concluding Remarks 45

Acknowledgements 46

References 46

3 Resonance Assignments 55
Lu-Yun Lian and Igor L. Barsukov

3.1 Introduction 55

3.2 Resonance Assignment of Unlabelled Proteins 56

3.2.1 Spin System Assignments 57

3.2.2 Sequence-Specific Assignments 59

3.2.3 Possible Difficulties 60

3.3 15 N-Edited Experiments 60

3.4 Triple Resonance 62

3.4.1 3D Triple Resonance 62

3.4.1.1 Identification of Spin Systems 64

3.4.1.2 Sequential Assignment 68

3.4.1.3 Proline Residues 74

3.4.2 4D Triple Resonance 74

3.4.3 Computer-Assisted Backbone Assignments 76

3.4.4 Unstructured Proteins 76

3.4.5 Large Proteins 77

3.5 Side-Chain Assignments 77

References 81

4 Measurement of Structural Restraints 83
Geerten W. Vuister, Nico Tjandra, Yang Shen, Alex Grishaev and Stephan Grzesiek

4.1 Introduction 83

4.2 NOE-Based Distance Restraints 84

4.2.1 Physical Background 84

4.2.2 NMR Experiments for Measuring the NOE 86

4.2.3 Set-up of NOESY Experiments 87

4.2.3.1 Estimation of T₂s 87

Recipe 4.1: 1-1 Echo Experiment 88

Recipe 4.2: Set-up of Optimal Acquisition Times 89

Recipe 4.3: Set-up of a 3D 15 N-Edited NOESY Experiment (Figure 4.2a) 90

Recipe 4.4: Set-up of a 3D 13 C-Edited NOESY Experiment 91

4.2.4 Deriving Structural Information from NOE Cross-peaks 92

Recipe 4.5: Extraction of Distances Using Classes 95

Recipe 4.6: Extraction of Distances Using the Two-Spin Approximation 95

4.2.5 Information Content of NOE Restraints 95

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