Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I OVERVIEW
1 Assessing and Improving Protein Sample Quality . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Bertrand Raynal, Se ´bastien Bru ˆ le ´, Stephan Uebel, and Stefan H. Knauer
2 A Familiar Protein–Ligand Interaction Revisited with Multiple Methods. . . . . . . 47
Xiaochun Li-Blatter, Ludovit Zweifel, and Timothy Sharpe
PART II UNIVERSAL METHODS FOR PROTEIN INTERACTIONS
3 Interactions of a Signal Transduction Protein Investigated by
Fluorescence Stopped-Flow Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Stephen R. Martin and Maria J. Schilstra
4 Kinetic Methods of Deducing Binding Mechanisms Involving
Intrinsically Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Elin Karlsson and Per Jemth
5 Isothermal Titration Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Christopher M. Johnson
6 Measuring the K D of Protein–Ligand Interactions Using
Microscale Thermophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Shih-Chia Tso and Chad A. Brautigam
7 Quartz Crystal Microbalance with Dissipation Monitoring
(QCM-D): Preparing Functionalized Lipid Layers for the Study
of Complex Protein–Ligand Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Holly L. Birchenough and Thomas A. Jowitt
PART III SCREENING FOR LIGAND BINDING
8 Indirect Detection of Ligand Binding by Thermal Melt Analysis . . . . . . . . . . . . . . 201
Joseph Shaw and Christopher Stubbs
9 The Use of Acoustic Mist Ionization Mass Spectrometry for
High-Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Helen Plant, David Murray, Hannah Semple, Gareth Davies,
Ian Sinclair, and Geoffrey A. Holdgate
10 Ligand Discovery: High-Throughput Binding: Fluorescence
Polarization (Anisotropy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Geoffrey A. Holdgate and Paul E. Hemsley
11 Fragment Screening by NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Ben J. Davis
vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I OVERVIEW
1 Assessing and Improving Protein Sample Quality . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Bertrand Raynal, Se ´bastien Bru ˆ le ´, Stephan Uebel, and Stefan H. Knauer
2 A Familiar Protein–Ligand Interaction Revisited with Multiple Methods. . . . . . . 47
Xiaochun Li-Blatter, Ludovit Zweifel, and Timothy Sharpe
PART II UNIVERSAL METHODS FOR PROTEIN INTERACTIONS
3 Interactions of a Signal Transduction Protein Investigated by
Fluorescence Stopped-Flow Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Stephen R. Martin and Maria J. Schilstra
4 Kinetic Methods of Deducing Binding Mechanisms Involving
Intrinsically Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Elin Karlsson and Per Jemth
5 Isothermal Titration Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Christopher M. Johnson
6 Measuring the K D of Protein–Ligand Interactions Using
Microscale Thermophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Shih-Chia Tso and Chad A. Brautigam
7 Quartz Crystal Microbalance with Dissipation Monitoring
(QCM-D): Preparing Functionalized Lipid Layers for the Study
of Complex Protein–Ligand Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Holly L. Birchenough and Thomas A. Jowitt
PART III SCREENING FOR LIGAND BINDING
8 Indirect Detection of Ligand Binding by Thermal Melt Analysis . . . . . . . . . . . . . . 201
Joseph Shaw and Christopher Stubbs
9 The Use of Acoustic Mist Ionization Mass Spectrometry for
High-Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Helen Plant, David Murray, Hannah Semple, Gareth Davies,
Ian Sinclair, and Geoffrey A. Holdgate
10 Ligand Discovery: High-Throughput Binding: Fluorescence
Polarization (Anisotropy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Geoffrey A. Holdgate and Paul E. Hemsley
11 Fragment Screening by NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Ben J. Davis
vii