Biothermodynamics, Part C

1. Auflage 2010
ISBN: 978-0-12-381269-8
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 488, 401 Seiten, Web PDF

Reihe: Methods in Enzymology

In the past several years, there has been an explosion in the ability of biologists, molecular biologists and biochemists to collect vast amounts of data on their systems. Biothermodynamics, Part C presents sophisticated methods for estimating the thermodynamic parameters of specific protein-protein, protein-DNA and small molecule interactions. The use of thermodynamics in biological research is used as an 'energy book-keeping system. While the structure and function of a molecule is important, it is equally important to know what drives the energy force. These methods look to answer: What are the sources of energy that drive the function? Which of the pathways are of biological significance? As the base of macromolecular structures continues to expand through powerful techniques of molecular biology, such as X-ray crystal data and spectroscopy methods, the importance of tested and reliable methods for answering these questions will continue to expand as well. - Elucidates the relationships between structure and energetics and their applications to molecular design, aiding researchers in the design of medically important molecules - Provides a 'must-have' methods volume that keeps MIE buyers and online subscribers up-to-date with the latest research - Offers step-by-step lab instructions, including necessary equipment, from a global research community

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology: Biothermodynamics, Part C;4
3;Copyright Page;5
4;Contents;6
5;Contributors;12
6;Preface;16
7;Methods in Enzymology;18
8;Chapter 1: Measurement and Analysis of Equilibrium Binding Titrations: A Beginner’s Guide;48
8.1;1. Material Requirements for Binding Measurements;49
8.2;2. Monitoring a Binding Reaction;49
8.3;3. The Binding Equation and Its Relationship to Binding Measurements ;52
8.4;4. Plotting and Analysis of Binding Data;55
8.5;5. Protein Concentration Is Important: Equilibrium Versus Stoichiometric Conditions;57
8.6;6. When Are Total and Free Ligand Concentrations Equal?;61
8.7;7. Deviations from Simple Binding;61
8.8;References;63
9;Chapter 2: Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs;64
9.1;1. Introduction;65
9.2;2. A Single Titration Curve: Some Simple Considerations of Possible Pitfalls;67
9.3;3. Quantitative Equilibrium Spectroscopic Titrations: Thermodynamic Bases;71
9.4;4. Nucleotide Binding to the RepA Protein of Plasmid RSF1010;76
9.5;5. Applying the Statistical Thermodynamic Model for the Nucleotide Binding to the RSF1010 RepA Protein Hexamer;79
9.6;6. Empirical Function Approach;82
9.7;7. MCT Method: General Considerations ;83
9.8;8. Application of the MCT Method to the Base Specificity Problem in ASFV Pol X-ssDNA System;86
9.9;9. Application of MCT Method to Protein-ssDNA Lattice Binding Systems;88
9.10;10. Quantitative Analysis of the Binding of the E. coli DnaB Helicase to Unmodified Nucleic Acids Using the MCT Method;93
9.11;11. Direct Analysis of the Experimental Isotherm of Protein Ligand Binding to Two Competing Nucleic Acid Lattices;95
9.12;12. Using a Single Concentration of a Nonfluorescent Unmodified Nucleic Acid;99
9.13;13. Using Short Fluorescent Oligonucleotides in Competition with the Polymer Nucleic Acid;100
9.14;14. Conclusions;102
9.15;Acknowledgments;102
9.16;References;103
10;Chapter 3: Analysis of PKR-RNA Interactions by Sedimentation Velocity;106
10.1;1. Introduction;107
10.2;2. Reagents and Cells;109
10.3;3. Experimental Design;111
10.4;4. Examples;114
10.5;5. Conclusions;122
10.6;Acknowledgments;122
10.7;References;123
11;Chapter 4: Structural and Thermodynamic Analysis of PDZ-Ligand Interactions;128
11.1;1. Introduction;129
11.2;2. Structural Studies of the Tiam1 PDZ Domain;130
11.3;3. Fluorescence Anisotropy Methods for Measuring the Energetics of PDZ-Ligand Interactions;134
11.4;4. Double-Mutant Cycle Analysis of PDZ-Binding Pockets;140
11.5;5. Peptide Evolution as a Tool for Probing PDZ Specificity;142
11.6;6. Conclusions;145
11.7;Acknowledgments;146
11.8;References;146
12;Chapter 5: Thermodynamic Analysis of Metal Ion-Induced Protein Assembly;148
12.1;1. Introduction;149
12.2;2. Linked Equilibria-General Concepts;150
12.3;3. Experimental Approaches-Analytical Ultracentrifugation;153
12.4;4. Summary;165
12.5;Acknowledgments;166
12.6;References;166
13;Chapter 6: Thermodynamic Dissection of Colicin Interactions;170
13.1;1. Introduction;171
13.2;2. DNase Domain-Immunity Protein Interactions;179
13.3;3. Receptor Binding;184
13.4;4. Mapping Binding Epitopes and Signaling Networks;187
13.5;5. Discussion;190
13.6;References;191
14;Chapter 7: Energetics of Src Homology Domain Interactions in Receptor Tyrosine Kinase-Mediated Signaling;194
14.1;1. Introduction;195
14.2;2. Interactions of Src Homology 2 Domains;196
14.3;3. Recognition by the "Two-Pinned Plug" ;200
14.4;4. Recognition by the beta-Turn Motif;206
14.5;5. Selectivity Versus Specificity for SH2 Domain Interactions;207
14.6;6. Proline Sequence-Recognition Domains;209
14.7;7. Interactions of SH3 Domains;210
14.8;8. What Constitutes Specificity in SH3 Domain Interactions?;216
14.9;9. Selectivity in SH3 Domain Interactions;218
14.10;10. Interactions Through Multiple Domains;221
14.11;11. Conclusions;223
14.12;References;224
15;Chapter 8: Structural and Functional Energetic Linkages in Allosteric Regulation of Muscle Pyruvate Kinase;232
15.1;1. Introduction;234
15.2;2. General Principles of Linked Multiequilibria Reactions;234
15.3;3. Functional Energetic Linkages in Allosteric Regulation of Rabbit Muscle Pyruvate Kinase;236
15.4;4. Functional Linkage Through Steady-State Kinetics;237
15.5;5. Structural Perturbations by Ligands;240
15.6;6. Functional Linkage Scheme of Allostery for RMPK;248
15.7;7. Functional Linkage Through Ligand Binding Measurements ;249
15.8;8. Protein Structural Dynamics-Amide Hydrogen Exchange Monitored by FT-IR (HX-FT-IR) ;256
15.9;9. Probing Interfacial Interactions;257
15.10;10. Summary Statement;259
15.11;Acknowledgments;260
15.12;References;260
16;Chapter 9: Analysis of Free Energy Versus Temperature Curves in Protein Folding and Macromolecular Interactions;266
16.1;1. Stability Curves=Gibbs-Helmholtz Curves=DeltaG Versus Temperature;267
16.2;2. Analysis of DeltaG Versus Temperature in Protein Folding;270
16.3;3. Using Stability Curves to Compare Mesophilic and Thermophilic Protein Pairs;272
16.4;4. Temperature Dependence of Folding Enthalpies and Entropies;273
16.5;5. Analysis of DeltaG Versus Temperature Data in Macromolecular Interactions;276
16.6;6. Fitting DeltaH and DeltaG Versus Temperature for a DeltaDeltaCp;277
16.7;7. Examples of Potential Consequences of a Small DeltaDeltaCp;282
16.8;References;284
17;Chapter 10: Application of the Sequential n-Step Kinetic Mechanism to Polypeptide Translocases;286
17.1;1. Introduction;287
17.2;2. Single-Turnover Fluorescence Stopped-Flow Method to Monitor Polypeptide Translocation;288
17.3;3. Application of the Sequential n-Step Mechanism;293
17.4;4. Concluding Remarks;309
17.5;Acknowledgments;310
17.6;References;310
18;Chapter 11: A Coupled Equilibrium Approach to Study Nucleosome Thermodynamics;312
18.1;1. Introduction;313
18.2;2. Salt-Mediated Nucleosome (Dis)Assembly;314
18.3;3. A Chaperone-Mediated Coupled Approach to Nucleosome Thermodynamics ;316
18.4;4. Experimental Setup and Considerations;322
18.5;5. Data Analysis and Theory;323
18.6;6. Summary and Implications;329
18.7;References;331
19;Chapter 12: Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo;334
19.1;1. Introduction;335
19.2;2. DNA Polymer Theory;336
19.3;3. Ligase-Catalyzed DNA Cyclization Kinetics In Vitro;339
19.4;4. In Vivo Analysis of E. coli lac Repression Loops;349
19.5;Acknowledgments;361
19.6;Appendix A. R Code for j-Factor Experiments;361
19.7;Appendix B. R Code for lac Looping Experiments;366
19.8;Appendix C. Required Files for R scripts ;379
19.9;References;380
20;Author Index;384
21;Subject Index;396
22;Color Plates;402