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The combination of faster, more advanced computers and more quantitatively oriented biomedical researchers has recently yielded new and more precise methods for the analysis of bi… Read more
SUSTAINABLE DEVELOPMENT
Save up to 30% on top Physical Sciences & Engineering titles!
The combination of faster, more advanced computers and more quantitatively oriented biomedical researchers has recently yielded new and more precise methods for the analysis of biomedical data. These better analyses have enhanced the conclusions that can be drawn from biomedical data, and they have changed the way that experiments are designed and performed. This volume, along with the 2 previous Computer Methods volumes for the Methods in Enzymology serial, aims to inform biomedical researchers about recent applications of modern data analysis and simulation methods as applied to biomedical research.
Biochemists, molecular biologists, cell biologists, biomedical researchers, microbiologists, and developmental biologists
Preface
Measurement and Analysis of Equilibrium Binding Titrations
1. Material Requirements for Binding Measurements
2. Monitoring a Binding Reaction
3. The Binding Equation and Its Relationship to Binding Measurements
4. Plotting and Analysis of Binding Data
5. Protein Concentration Is Important: Equilibrium Versus Stoichiometric Conditions
6. When Are Total and Free Ligand Concentrations Equal?
7. Deviations from Simple Binding
Macromolecular Competition Titration Method
1. Introduction
2. A Single Titration Curve: Some Simple Considerations of Possible Pitfalls
3. Quantitative Equilibrium Spectroscopic Titrations: Thermodynamic Bases
4. Nucleotide Binding to the RepA Protein of Plasmid RSF1010
5. Applying the Statistical Thermodynamic Model for the Nucleotide Binding to the RSF1010 RepA Protein Hexamer
6. Empirical Function Approach
7. MCT Method: General Considerations
8. Application of the MCT Method to the Base Specificity Problem in ASFV Pol X–ssDNA System
9. Application of MCT Method to Protein–ssDNA Lattice Binding Systems
10. Quantitative Analysis of the Binding of the E. coli DnaB Helicase to Unmodified Nucleic Acids Using the MCT Method
11. Direct Analysis of the Experimental Isotherm of Protein Ligand Binding to Two Competing Nucleic Acid Lattices
12. Using a Single Concentration of a Nonfluorescent Unmodified Nucleic Acid
13. Using Short Fluorescent Oligonucleotides in Competition with the Polymer Nucleic Acid
14. Conclusions
Acknowledgments
Analysis of PKR–RNA Interactions by Sedimentation Velocity
1. Introduction
2. Reagents and Cells
3. Experimental Design
4. Examples
5. Conclusions
Acknowledgments
Structural and Thermodynamic Analysis of PDZ–Ligand Interactions
Abbreviations
1. Introduction
2. Structural Studies of the Tiam1 PDZ Domain
3. Fluorescence Anisotropy Methods for Measuring the Energetics of PDZ–Ligand Interactions
4. Double-Mutant Cycle Analysis of PDZ-Binding Pockets
5. Peptide Evolution as a Tool for Probing PDZ Specificity
6. Conclusions
Acknowledgments
Thermodynamic Analysis of Metal Ion-Induced Protein Assembly
1. Introduction
2. Linked Equilibria—General Concepts
3. Experimental Approaches—Analytical Ultracentrifugation
4. Summary
Acknowledgments
Thermodynamic Dissection of Colicin Interactions
1. Introduction
2. DNase Domain–Immunity Protein Interactions
3. Receptor Binding
4. Mapping Binding Epitopes and Signaling Networks
5. Discussion
Energetics of Src Homology Domain Interactions in Receptor Tyrosine Kinase-Mediated Signaling
1. Introduction
2. Interactions of Src Homology 2 Domains
3. Recognition by the “Two-Pinned Plug”
4. Recognition by the β-Turn Motif
5. Selectivity Versus Specificity for SH2 Domain Interactions
6. Proline Sequence-Recognition Domains
7. Interactions of SH3 Domains
8. What Constitutes Specificity in SH3 Domain Interactions?
9. Selectivity in SH3 Domain Interactions
10. Interactions Through Multiple Domains
11. Conclusions
Structural and Functional Energetic Linkages in Allosteric Regulation of Muscle Pyruvate Kinase
1. Introduction
2. General Principles of Linked Multiequilibria Reactions
3. Functional Energetic Linkages in Allosteric Regulation of Rabbit Muscle Pyruvate Kinase
4. Functional Linkage Through Steady-State Kinetics
5. Structural Perturbations by Ligands
6. Functional Linkage Scheme of Allostery for RMPK
7. Functional Linkage Through Ligand Binding Measurements
8. Protein Structural Dynamics—Amide Hydrogen Exchange Monitored by FT-IR (HX-FT-IR)
9. Probing Interfacial Interactions
10. Summary Statement
Acknowledgments
Analysis of Free Energy Versus Temperature Curves in Protein Folding and Macromolecular Interactions
1. Stability Curves=Gibbs–Helmholtz Curves=ΔG Versus Temperature
2. Analysis of ΔG Versus Temperature in Protein Folding
3. Using Stability Curves to Compare Mesophilic and Thermophilic Protein Pairs
4. Temperature Dependence of Folding Enthalpies and Entropies
5. Analysis of ΔG Versus Temperature Data in Macromolecular Interactions
6. Fitting ΔH and ΔG Versus Temperature for a ΔΔCp
7. Examples of Potential Consequences of a Small ΔΔCp
Application of the Sequential n-Step Kinetic Mechanism to Polypeptide Translocases
1. Introduction
2. Single-Turnover Fluorescence Stopped-Flow Method to Monitor Polypeptide Translocation
3. Application of the Sequential n-Step Mechanism
4. Concluding Remarks
Acknowledgments
A Coupled Equilibrium Approach to Study Nucleosome Thermodynamics
1. Introduction
2. Salt-Mediated Nucleosome (Dis)Assembly
3. A Chaperone-Mediated Coupled Approach to Nucleosome Thermodynamics
4. Experimental Setup and Considerations
5. Data Analysis and Theory
6. Summary and Implications
Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo
1. Introduction
2. DNA Polymer Theory
3. Ligase-Catalyzed DNA Cyclization Kinetics In Vitro
4. In Vivo Analysis of E. coli lac Repression Loops
Acknowledgments
Appendix A. R Code for j-Factor Experiments
Appendix B. R Code for lac Looping Experiments
Appendix C. Required Files for R scripts
MS