Ligand-Binding Basics - Evaluating Intermolecular Affinity, Specificity, Stoichiometry,

A concise and accessible textbook covering ligand-binding theory in chemistry, biology, and drug development
 
In Ligand-binding Basics: Evaluating Intermolecular Affinity, Specificity, Stoichiometry, and Cooperativity, accomplished chemist Jannette Carey introduces ligand binding in a thorough and practical way for those new to the topic, as well as anyone seeking a connection between theory and experiment. Using a minimum of mathematical formalism, this book offers analytical rigor while remaining accessible to non-specialist practitioners. It provides readers with the skills they need to analyze their own binding data or published results, helping them develop an intuitive grasp of ligand-binding phenomena integrated with structural and thermodynamic understanding.
 
Topics covered include:
* Application of the principles of equilibrium, mass action, and mass balance to derive the basic equations that describe all binding processes
* Recommended approaches for plotting and graphical analysis of binding data
* Strategies for designing, analyzing, interpreting, and troubleshooting experiments from the perspective of ligand-binding theory
* Review of selected examples that illustrate integration of structural and thermodynamic analysis
 
Perfect for students and educators in chemistry, biochemistry, molecular biology, and pharmaceutical science, Ligand-binding Basics will also appeal to practitioners who aim to study ligand binding in any molecular system.

List of contents

About the Cover xi
Introduction xiii
1 The Biology of Molecules 1
Why Study Intermolecular Interactions Quantitatively? 1
Equilibrium and Kinetics 2
Thermodynamic Definitions of Affinity and Specificity 3
The Affinity/Specificity Map 6
Biology Requires Optimization of Affinity and Specificity 8
The Special Case of Protein-DNA Interactions 8
2 General Theory for Reversible Ligand Binding 10
Definition of Ligand and Titration 10
Affinity, Specificity, Stoichiometry, and Cooperativity 10
Ligand-binding Theory: Relationship to Experiment 13
General Theory for Reversible Ligand Binding: Rooted in Chemical Equilibrium 14
General Theory for Reversible Ligand Binding: Quantitative Treatment 14
The Case of 1:1 Binding 15
General Theory for Reversible Ligand Binding: Conservation of Mass 17
Definition of ¿ 18
The Basic Equation for 1:1 Binding 19
The Single Most Important Thing You Can Learn in This Book 20
The Example of Heme Binding to Apocytochrome c 21
The Rectangular Hyperbola 22
The Binding Isotherm 23
Plot of ¿ vs. [H f ] 24
General Theory for Reversible Ligand Binding: Role of Mass Action 25
Plot of [AH]vs.[H t ] with Fixed K 27
Determination of K d from Experiment 28
Plot of [AH]vs.[H t ] with Fixed [A t ] 29
Determining Molar Ratio from Experiment 29
About Activity 31
3 Graphical Analysis 33
Limitations of Direct Plots 33
The Semi-log Plot 34
Breadth of the Semi-log Plot 36
Myoglobin and Hemoglobin 38
Advantages of the Direct and Semi-log Plots of Binding Data 40
Linear Transforms of the Basic Binding Equation 40
Common Linearizations 41
Requirements of the Linear Regression Model 41
A Linear Model May Misrepresent the Physical Process 43
Deviations from Linearity Are Hard to Detect or Interpret 44
Linear Transforms Distort Data Completeness 44
Linear Transforms Invite - Even Require - Extrapolation 46
Linear Transforms Falsely Promise Both K and Molar Ratio from a Single Dataset 47
Summary about Linear Treatments of Binding Data 47
Simulation Is Just as Good as Fitting, Given Realistic Experimental Errors 50
4 Binding of Multiple Ligands 52
Conservation of Mass Outside the 1:1 Case 52
Redefine ¿ to Accommodate Any Molar Ratio 53
Accounting for the Definition of Molecule 54
Generalizing to Integer Multiples of 1:1 54
The Langmuir Equation for Any Molar Ratio with Sites of Identical Affinity and No Cooperativity 56
Adair Equation for Any Number of Binding Events 57
The Langmuir Equation vs. the Adair Equation 60
Thermodynamic Linkage 61
Two Classes of Sites with Different Affinities 62
Binding Isotherms for Multiple Sites with Different Affinities 62
Summary 66
5 How to Determine K d and Molar Ratio Experimentally 67
Stoichiometric Titration First 68
Amounts of Materials 69
Assigning Partners 69
Choice of Experimental Observables 70
Choosing Solution Conditions 70
How Many Data Points? 71
Range-Finding Stoichiometric Titration 72
Visualizing Results 73
Range-Finding Asymptotic Titration to Estimate K d 74
Data Analysis 75
Practicalities about Experimental Error 75
Statistical Approaches to Estimate the Breakpoint 76
Refined Asymptotic Titration 76
Designing an Experiment to Refine K d 77
Calculating Free Ligand Concentration 78
Refining the Value of Molar Ratio 79
Example of ArgR/DNA Binding 79
Plotting the Data 81
Deriving K d from the Data 81
Summary 81
6 Cooperativity 83
Facilitated and Antagonized Binding 83
Free Energy Definition of Cooperative Binding 84
Chemical Potential Diagram for Cooperative Binding 86
Cooperativity as Non-additivity 87
Reciprocity of Cooperative Effects 88
Limitations of Linear Transforms for Cooperative Interactions 88
Microscopic View of Species Distribution 89
Homotropic and Heterotropic Cooperativity 90
Cooperativity Affects Specificity as Well as Affinity 92
Cooperativity Is the Third Axis of the Affinity/Specificity Map 94
Quantifying Homotropic Cooperativity 95
Negative Homotropic Cooperativity 95
A Practical Advantage of Negative Cooperativity 97
Positive Cooperativity and the Ligand Concentration Interval 97
Importance of Individual-site Isotherms and Species Distribution 100
Species Distributions by Specialized Experimental Methods 101
The Many Forms of Cooperativity 103
Emergent Properties 103
Connectivity and Search Entropy 104
Breakdown of Additivity in Complex Systems 105
Statistical Effects 107
Relevance of Non-additivity for Analysis of Mutations 110
Universality and Promiscuity of Cooperativity 111
Proteins as Gestalt Objects 113
Summary 115
7 Theoretical and Method-specific Troubleshooting 116
Equilibrium and Nonequilibrium Methods 116
Accessible Concentration Ranges Limit Accessible K d Values 116
Signal from Ligand or Target? 118
Separation-based Methods 118
Filter Binding 119
Gel Retardation or EMSA 120
Gel Filtration 121
Hummel and Dreyer Chromatography 121
Equilibrium Dialysis 122
UV Absorbance 123
CD Spectroscopy 123
Fluorescence 124
NMR 124
ITC 125
AUC 129
SPR 129
MS 131
8 Allostery 133
An Historical Overview 133
Facilitated Binding 135
Elaboration of the MWC Model 136
Relaxed Monomers and Tense Multimers 136
Positive Homotropic Cooperativity Only 137
Artifactual Origins of Affinity Heterogeneity 138
Relaxation of Multimers by Ligand Binding 138
Koshland's Sequential (Asymmetric) Model 140
G3Pase Was Heterogeneous, Not Negatively Cooperative 141
Many Models Fit the Hemoglobin Data 142
Advantages of Negative Cooperativity for Molecular Insight 143
Biology of Negative Cooperativity 145
Structural Analysis Cannot Solve Allostery 146
Allostery without Cooperativity 147
Summary 148
9 Lessons on Affinity and Specificity from Host/Guest Chemistry 149
2D Representations of 3D Objects 149
Early Hosts Were Linear and Flexible 150
Design of Molecular Properties 151
Very Weak Affinity and No Detectable Specificity 151
Later Hosts Pre-organized in Bound Conformation 152
Enormous Gains in Affinity and Specificity 152
Bonds between Host and Guest Are Identical 153
Lessons from the Host/Guest Chemistry 153
Rational Design of Affinity and Specificity 153
Affinity and Specificity Accrue in Parallel 155
Cryptic Contributions Can Dominate Binding 156
10 Reconciling Structure and Thermodynamics in Molecular Interactions 157
Thermodynamics of Molecular Interactions 158
Structural Analysis of Bonding Does Not Predict Binding 160
The Goldilocks Region of Affinity/Specificity Space 162
Conformational Rearrangement upon Binding Decouples Affinity and Specificity 163
A Reservoir of Adaptability 164
No Simple Reconciliation of Structural and Energetic Views 165
Implications for Drug Design 166
11 Applications in Modern Drug Development 167
Background 167
Technological Developments 167
Crystal Structures 168
Trapped High-energy States 168
Another Example 171
Computational Methods 175
High-throughput Assays 177
Druggability 178
Irrational Drug Design 180
A New Workflow 181
Appendix A Ligand-binding Study Questions 182
Appendix B Thought Experiments 195
Appendix C Derivations 197
Appendix D Simulation and Fitting 201
Simulation 201
Fitting 203
Appendix E About the Hill Equation 208
Deriving the Hill Equation 208
The Hill Equation as a Limit of the Adair Equation 209
On Applying the Hill Equation to Quantify Cooperativity 210
Appendix F Stereo Viewing 212
Bibliography 215
Index 227

About the author

Jannette Carey has been a member of the Chemistry faculty at Princeton University for over thirty years, where she developed and teaches a two-term sequence in biophysical chemistry that is accessible to early graduate and advanced undergraduate students in a wide range of disciplines. Her biophysical research is focused on unifying the thermodynamic and structural basis for macromolecular interactions.

Summary

A concise and accessible textbook covering ligand-binding theory in chemistry, biology, and drug development

In Ligand-binding Basics: Evaluating Intermolecular Affinity, Specificity, Stoichiometry, and Cooperativity, accomplished chemist Jannette Carey introduces ligand binding in a thorough and practical way for those new to the topic, as well as anyone seeking a connection between theory and experiment. Using a minimum of mathematical formalism, this book offers analytical rigor while remaining accessible to non-specialist practitioners. It provides readers with the skills they need to analyze their own binding data or published results, helping them develop an intuitive grasp of ligand-binding phenomena integrated with structural and thermodynamic understanding.

Topics covered include:
* Application of the principles of equilibrium, mass action, and mass balance to derive the basic equations that describe all binding processes
* Recommended approaches for plotting and graphical analysis of binding data
* Strategies for designing, analyzing, interpreting, and troubleshooting experiments from the perspective of ligand-binding theory
* Review of selected examples that illustrate integration of structural and thermodynamic analysis

Perfect for students and educators in chemistry, biochemistry, molecular biology, and pharmaceutical science, Ligand-binding Basics will also appeal to practitioners who aim to study ligand binding in any molecular system.

Product details

• Authors: J Carey, Jannette Carey, Jannette (Princeton University Carey, Jannette (Princeton University) Carey
• Assisted by: Ethan Sample (Drawings)
• Publisher: Wiley, John and Sons Ltd
• Languages: English
• Product format: Hardback
• Released: 01.06.2024
• EAN: 9781119878421
• ISBN: 978-1-119-87842-1
• No. of pages: 256
• Subjects: Natural sciences, medicine, IT, technology > Chemistry; Chemie, Biochemie, Physikalische Chemie, Proteine, Life Sciences, Biowissenschaften, chemistry, Physical Chemistry, Biochemie u. Chemische Biologie, Biochemistry (Chemical Biology), Proteins & Macromolecules, Proteine u. Makromoleküle