Protein-Ligand Interactions: hydrodynamics and calorimetry

Transcription

1 Protein-Ligand Interactions: hydrodynamics and calorimetry Approach Stephen E. Harding Babur Z. Chowdhry OXFORD UNIVERSITY PRESS

2 , New York Oxford University Press,

3 List of protocols page Abbreviations xxi xvii 1 Protein-ligand interactions and their analysis Babur Z. Chowdhry and Stephen E. Harding 1 Introduction 1 2 A brief comparison of the 'high resolution' methods 6 3 Other spectroscopic techniques 12 4 Non-spectral methods 13 5 Computational methods/molecular modelling 14 6 Information sources 16 7 Additional remarks: the layout of the volumes 16 References 17 2 Equilibrium dialysis and rate dialysis 19 Bent Honore 1 Introduction 19 2 Equilibrium dialysis 21 Principle 21 Measurement of free ligand by UV or visible light absorption 21 Running an equilibrium dialysis experiment 23 Running control experiments for equilibrium dialysis 27 Measurement of ligand concentration by using a radiolabelled ligand 31 3 Rate dialysis 34 Principle and theory 34 Running experiments using rate dialysis 37 Rate dialysis in microchambers 40 4 Rate dialysis for measurement of free ligand concentrations in complex mixtures, e.g. serum 41 Acknowledgements 45 References 45

4 3 Quantitative characterization of ligand binding by chromatography 47 Donald J. Winzor 1 Introduction 47 2 General experimental aspects 48 Zonal and frontal affinity chromatography techniques 48 Comparison of advancing and trailing elution profiles: the concept of nonenantiography 48 Elution volume: an equilibrium parameter 50 Measurement of binding constants 51 3 Frontal gel chromatographic studies of ligand binding 52 Evaluation of the binding constant for a 1:1 interaction 52 Interaction of a univalent ligand with a multivalent acceptor 56 Allowance for multivalence of the ligand 57 Evaluation of K AS from the dependence of the constituent elution volume of acceptor upon ligand concentration 59 4 Zonal gel chromatographic studies of ligand binding 61 The Hummel and Dreyer technique 61 A limitation of the Hummel and Dreyer technique 62 5 Evaluation of binding constants by quantitative affinity chromatography 63 General theoretical aspects 64 Evaluation of ligand binding constants by frontal affinity chromatography 66 Zonal quantitative affinity chromatography 68 Other forms of quantitative affinity chromatography 70 6 Concluding remarks 72 References 72 4 Sedimentation velocity analytical ultracentrifugation 75 Stephen E. Harding and Donald J. Winzor 1 Introduction: sedimentation velocity and sedimentation equilibrium 75 2 Basic principles of sedimentation velocity 76 Measurement of a sedimentation coefficient 76 Measurement of molecular mass by sedimentation velocity 78 3 General experimental aspects 82 Optical systems for sedimentation velocity and sedimentation equilibrium 82 Sedimentation velocity optical records 83 Data capture 84 Two complications 85 Co-sedimentation diagrams 85 Concentration dependence of the sedimentation coefficient 85 Sedimentation coefficient ratios 88 Sedimentation velocity fingerprinting 89 4 Sedimentation velocity analysis of the shape of a molecular complex 89 Direct evaluation of molecular shape 90 Selecting a plausible structure which best agrees with the data 90

5 5 Sedimentation velocity studies of ligand binding 91 Interactions of a (protein) acceptor with a small ligand 91 Interactions of an acceptor with a macromolecular ligand 92 Sedimentation velocity studies of weak interactions 94 The shape of sedimenting boundaries for acceptor-ligand systems 95 6 The study of ligand-mediated conformational changes 98 7 Concluding remarks 101 References Sedimentation equilibrium in the analytical ultracentrifuge 1os Donald J. Winzor and Stephen E. Harding 1 Introduction Experimental aspects of sedimentation equilibrium 106 Procedural details of a sedimentation equilibrium experiment 107 Extraction of the molecular weight of a single solute 109 Extraction of point average molecular weights for interacting systems 115 Direct curve-fitting of concentration distributions 116 Omega and psi analyses of sedimentation equilibrium distributions Sedimentation equilibrium studies of ligand binding 121 Evaluation of the concentration distributions of individual species 121 Direct modelling of sedimentation equilibrium distributions Ligand perturbation of acceptor self-association 126 Characterization of acceptor self-association by sedimentation equilibrium 126 Displacement of an acceptor self-association equilibrium by ligand binding 130 Illustrative studies of preferential ligand binding by sedimentation equilibrium Concluding remarks 132 References Surface plasmon resonance 137 P. Anton Van Der Merwe 1 Introduction Principles and applications of surface plasmon resonance 137 Principles 137 Applications General principles of BlAcore experiments 141 A typical experiment 141 Preparation of materials and buffers 141 Monitoring the dips Ligand 143 Direct versus indirect immobilization 143 Covalent immobilization 143 Non-covalent immobilization (ligand capture) 149 Activity of immobilized ligand 152 Control surfaces 152 Reusing sensor chips 153

6 5 Analyte 153 Purity, activity, and concentration 153 Valency 153 Refractive index effect and control analytes 154 Low molecular weight analytes Qualitative analysis: do they interact? 155 Positive and negative controls 155 Qualitative comparisons using a multivalent analyte Quantitative measurements 155 Affinity 156 Kinetics 160 Stoichiometry 165 Thermodynamics 165 Activation energy Conclusion Appendix 167 Physical basis of SPR 167 Samples and buffers 168 Additional information 168 Acknowledgements 169 References Capillary electrophoresis 171 Niels H. H. Heegaard 1 Introduction The capillary electrophoresis technique Why use electrophoresis for the study of reversibly interacting molecules? Instrumentation and experimental variables 175 Apparatus 175 Experimental variables Capillary electrophoretic binding experiments 181 Experimental approaches for electrophoretic binding studies 181 Demonstration of ligand binding 182 Determination of binding constants Conclusions 192 Acknowledgements 193 References Molecular electro-optics 197 Dietmar Porschke 1 Introduction Equations for representing the field-induced orientation of molecules 200 Dichroism amplitude 200 Dichroism decay 201

7 3 Instruments 203 General 203 Pulse generators 204 Measuring cells 205 Spectrophotometric detection 205 Transient data storage and data processing 207 Automatic data collection Experimental procedures 207 Sample preparation 207 Measurements Data evaluation 211 Amplitudes: the stationary dichroism 211 Interpretation 214 Simulations of electro-optical data from macromolecular structures Various spectroscopic techniques 216 Birefringence 216 Fluorescence 218 Light scattering Kinetics 219 References High-flux X-ray and neutron solution scattering 223 Stephen J. Perkins 1 Introduction Applications of scattering 226 Complementary structural approaches 226 Properties of X-ray scattering 227 Properties of neutron scattering X-ray and neutron facilities 229 High flux sources 229 X-ray instrumentation 232 Neutron reactor instrumentation 234 Spallation neutron instrumentation Experimental approach 238 Applications for X-ray and neutron beamtime 238 Preparation of protein-ligand complexes for scattering 239 Data collection strategies 242 Guinier analyses of the reduced scattering curve 248 Distance distribution function analyses Structural modelling of scattering data 252 Sphere modelling of scattering curves: use of atomic structures 252 Spherical harmonics modelling 255 Other modelling using spheres, shells, or ellipsoids 255 Comparison with sedimentation and diffusion coefficients Conclusions and worked examples 256 Small ligand binding to a protein (AmiC) 257 Ligand binding to a homodimeric complex (serum amyloid P component) 258

8 A heterodimeric complex (factor Vila-tissue factor complex) 259 A protein-dna complex (RuvA-Holliday junction complex) 259 Acknowledgements 260 References Isothermal titration calorimetry of biomolecules 263 Ronan O'Brien, Babur Z. Chowdhry, and John E. Ladbury 1 Introduction Principle of operation 264 Instrumentation 264 Instrumental calibration Full thermodynamic characterization of biological interactions 268 Derivation of thermodynamic parameters 268 Heats of ionization and ph dependence 269 Determination of AG and AS 272 Determination of the change in heat capacity Preparation for an ITC experiment 273 Concentration requirements 273 Sample preparation 273 Use of mixed solvent systems 274 Concentration determination ITC experimental protocol 276 Performing a titration 276 Trouble-shooting 277 Controls ITC data handling 278 Data fitting 278 Data analysis 279 Choosing the appropriate model Structure/thermodynamic relationships 281 Heat capacity versus buried surface area 281 Thermodynamic insights into the mechanisms of interaction involving DNA Extending the range of binding constants determination Advantages and disadvantages 283 References Differential scanning microcalorimetry 287 Alan Cooper, Margaret A. Nutley, and Abdul Wadood 1 Introduction DSC basics 288 Instrumentation 287 Quantitative analysis of DSC data-practical considerations 290 Concentration measurements 293 Units 293 Scan rates/reversibility 294 The baseline problem 294

9 3 Thermodynamic background 297 Heat capacity, enthalpy, and entropy 297 Equilibrium and free energy 299 Temperature dependence of thermodynamic quantities 299 The van't Hoff enthalpy Effects of ligand binding 302 Ligand binding and folding equilibrium 303 Change in T m 307 Effects when ligand binds to both N and U 308 One peak or two? 308 Hydrogen ions as ligands: the effect of ph on protein stability 313 Non-specific metal ion effects 315 Acknowledgements 317 References 318 List of suppliers 319 index 327