Introduction to Macromolecular Binding Equilibria / Edition 1

by Charles P. Woodbury

Overview

Macromolecules in the body form noncovalent associations, such as DNA-protein or protein-protein complexes, that control and regulate numerous cellular functions. Understanding how changes in the concentration and conformation of these macromolecules can trigger physiological responses is essential for researchers developing drug therapies to treat diseases affected by these imbalances.

Introduction to Macromolecular Binding Equilibria gives students in medicinal chemistry, pharmaceuticals, and bioengineering the necessary background in biophysical chemistry for research applications in drug discovery and development. Building upon a fundamental knowledge of calculus and physical chemistry, this compact, graduate-level text prepares students for advanced work in solution thermodynamics and binding phenomena and applying methods in this book to their own research.

This book describes the underlying theory of binding phenomena and explains how to apply the binding polynomial approach for building models and interpreting data. It also covers practical considerations for setting up binding experiments and describes how to obtain true thermodynamic isotherms unbiased by model assumption via model-free analysis of binding data.  

Product Details

ISBN-13:	9780367388324
Publisher:	Taylor & Francis
Publication date:	09/19/2019
Pages:	272
Product dimensions:	6.12(w) x 9.19(h) x (d)

About the Author

Woodbury, Charles P.

Table of Contents

Chapter 1 Binding Sites 1

1.1 The Importance and Complexity of Macromolecular Binding 1

1.1.1 Different Types of Multiple Equilibria in Macromolecular Binding 1

1.2 Generating Affinity and Specificity with Weak Interactions 4

1.2.1 Weak Interactions and Reversible Binding 4

1.2.2 Binding Specificity and Multiple Simultaneous Weak Interactions 5

1.2.3 The Strength of Binding 7

1.2.4 Enthalpy-Entropy Compensation 7

1.3 Size, Shape, and Functional Complementarity Determine Recognition 10

1.3.1 Exposed Surfaces and Binding 10

1.3.1.1 Accessible Surface Area 10

1.3.2 Convergence of Functional Groups 11

1.3.2.1 The Proximity or Chelate Effect 11

1.3.2.2 Clefts as a Structural Motif for Binding Sites 15

1.3.3 Conformational Flexibility 15

1.3.3.1 Microstates 15

1.3.3.2 Hydration and Flexibility 19

1.3.3.3 Time and Distance Scales 19

1.4 Binding Sites on Proteins 21

1.4.1 Macromolecular Structures and the Protein Data Bank 21

1.4.2 Small Molecule Sites 22

1.4.3 Protein-Protein Interfaces 23

1.4.4 Binding 'Hot Spots' 24

1.4.5 Protein Surfaces That Bind DNA 27

1.5 Binding Sites on Nucleic Acids 28

1.5.1 Nucleic Acids as Polyanions: Salt Effects in Ligand Binding 30

1.5.2 Nucleic Acid Double Helices: Contacts in the Grooves 30

1.5.3 Intercalative Binding 31

1.5.4 Sequence-Specific Binding 33

1.5.4.1 Sequence Recognition via the Major Groove 33

1.5.4.2 Site-Specific Binding in the Minor Groove 36

1.5.5 Nonspecific Binding and Ligand Sequestration 37

References 38

Chapter 2 Binding Isotherms 47

2.1 Some Definitions and Conventions on Notation 47

2.1.1 The Two Partners: Ligand and Macromolecule 47

2.1.2 Concentrations of Components 48

2.1.3 The Amount Bound: Binding Density and Degree of Saturation 48

2.1.4 Notation for Binding Constants 50

2.2 Connecting the Binding Density < r > with the Free Ligand Concentration [L] 51

2.2.1 Describing Binding at the Phenomenological Level 53

2.2.2 The Binding Isotherm as a Plot of < r > versus [L] 55

2.2.3 An Effective Binding Constant: The Concentration of Free Ligand at Half Saturation 56

2.2.4 The Question of Binding Stoichiometry 58

2.3 Simple Isotherm Models via the Binding Polynomial 59

2.3.1 Binding Free Energy Changes and the Binding Polynomial 59

2.3.2 The Langmuir Isotherm Model: Binding to Equal Independent Sites 61

2.3.3 Multiple Classes of Independent Sites 62

2.4 Graphical Methods 67

2.4.1 Virtues and Weaknesses of the Direct Plot 68

2.4.2 Linearized Plots 68

2.4.3 Some Common Errors of Experimental Design and Interpretation 71

2.4.3.1 Neglecting Corrections for Ligand Depletion and for Nonspecific Binding 71

2.4.3.2 Misinterpreting Slopes and Intercepts 73

References 77

Chapter 3 Binding Linkage, Binding Competition, and Multiple Ligand Species 79

3.1 The Binding Polynomial and Linked Binding Equilibria 81

3.1.1 Positive Linkage, Negative Linkage, and No Linkage between Species 81

3.1.2 Binding Competition 83

3.1.3 A Single Class of Binding Sites and Two Competing Ligand Species 84

3.1.3.1 Constant Concentration of One Species 84

3.1.3.2 Multiple Identical Sites 85

3.1.4 IC5Q Values and Competition Assays 86

3.1.4.1 Comparing Calcium Channel Blockers by Displacement Assay 87

3.1.5 Competitive Inhibition of an Enzyme 89

3.1.5.1 The Cheng-Prusoff Relations 89

3.1.5.2 Validating the Use of the Cheng-Prusoff Relations 91

3.1.6 Further Considerations in Competition Assays 91

3.2 Linkage and "Piggy-Back" Binding 92

3.2.1 Basic Theory for Piggy-Back Systems 93

3.2.2 Hydrogen Ion as a Piggy-Back Ligand: Theory for pH Effects 95

3.2.2.1 Titration of a Single Residue on the Ligand 95

3.2.2.2 Comparison to Titration of a Single Residue on the Receptor 97

3.2.3 pH Effects in RNase-Inhibitor Binding 97

3.3 Linkage Effects on Macromolecular Associations and Conformational Changes 98

3.3.1 Linkage and an A $$$ B Equilibrium 99

3.3.2 Ligand-Ligand Linkage and an A + B $$$ C Equilibrium 101

3.3.3 General Expression for the Salt Dependence of K_obs 102

3.3.4 Applications of Log-Log Plots 104

3.3.4.1 Salt Effects in α-Chymotrypsin Dimerization 105

3.3.4.2 Water Activity and Chloride Ion Binding in the Oxygenation of Hemoglobin 107

3.3.5 Uptake of L₂ by a Macromolecule Partially Saturated with L₁ 108

3.4 Linkage Involving Weak and Nonstoichiometric Binding 110

3.4.1 Preferential Interaction 111

3.4.2 Preferential Interaction and Macromolecular Equilibria 116

References 120

Chapter 4 Cooperativity 123

4.1 The Phenomenon of Binding Cooperativity 123

4.1.1 Cooperative Binding in the Oxygenation of Hemoglobin 123

4.1.2 Cooperativity in Enzyme Action 125

4.2 Terminology for Cooperative Interactions 127

4.3 Criteria for Cooperativity in Ligand Binding 128

4.3.1 Statistical Effects in Multisite Binding 129

4.3.2 The All-or-None Model and the Hill Plot 130

4.3.3 An Operational Definition for Cooperativity 135

4.3.4 Linkage Relations and Binding Cooperativity 138

4.4 Structural Models of Cooperative Binding 140

4.4.1 The Concerted Monod-Wyman-Changeux (MWC) Model 141

4.4.1.1 Heterotropic Effectors in the MWC Model 143

4.4.2 The Sequential Koshland-Nemethy-Filmer (KNF) Model 144

4.4.2.1 Heterotropic Effectors in the KNF Model 145

4.4.3 Comparison of the KNF and MWC Models 146

4.4.4 Oxygenation of Hemoglobin 148

4.4.5 Nesting 152

4.5 Aggregation and Cooperativity 153

4.5.1 Aggregation of Ligand as a Source of Binding Cooperativity 154

4.5.2 Aggregation of Receptor as a Source of Cooperativity 156

4.5.3 Ligand Dimerization Driven by a Piggy-Back Ligand 158

4.6 Negative Cooperativity 160

4.6.1 Negative Cooperativity in the Titration of Ethylene Diamine 162

4.6.2 Glyceraldehyde 3-Phosphate Dehydrogenase and Negative Cooperativity 163

References 166

Chapter 5 Binding to Lattices of Sites 171

5.1 Linear Lattices of Binding Sites 171

5.1.1 Redefining the Binding Density for Linear Systems 172

5.1.2 The McGhee-von Hippel Treatment [1] 175

5.1.3 Extensions of the Model: Oriented Lattices and Ligands 182

5.1.4 Site-Exclusion in DNA-Polyamine Binding 185

5.1.5 Site-Exclusion and Positive Cooperativity in DNA-Protein Binding 186

5.1.6 Site-Exclusion and Lattice Conformational Change 187

5.1.6.1 Ethidium/DNA Interactions 190

5.1.7 Piggy-Back Binding and DNA-Protein Interactions 192

5.2 Binding to Two-Dimensional Lattices 195

5.2.1 Heuristic Treatment of Site Exclusion in Two Dimensions 196

5.2.1.1 The Stankowski Model 196

5.2.2 Application to Membrane Binding 200

References 201

Chapter 6 Choosing a Method and Analyzing the Data 205

6.1 Considerations when Choosing a Method 205

6.1.1 Possible Assay Interference from Binding Kinetics 206

6.1.1.1 The Rate of Association 207

6.1.1.2 The Dissociation Rate 208

6.1.1.3 Further Kinetic Considerations 208

6.1.2 Direct versus Indirect Methods of Measuring the Equilibrium 209

6.2 Designing The Experiment 210

6.2.1 Nonideal Behavior: Salt and Crowding Effects 211

6.2.2 Choosing Working Concentrations in Relation to K 213

6.2.3 Some General Precautions to Consider 215

6.3 Model-Free Analyses of Binding Signals 216

6.3.1 Assumptions and Notation 217

6.3.2 Signal from the Ligand 218

6.3.3 Signal from the Macromolecule 220

6.4 Statistical Analysis of Binding Data 223

6.4.1 Errors in Dependent and Independent Variables 223

6.4.2 Computerized Fitting of Data 224

6.4.2.1 Nonlinear Curve Fitting 225

References 231

Appendix: The Sequence-Generating Function Method 235

Index 245