The use of thermodynamics in biological research can be equated to 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. This volume presents sophisticated methods for estimating the thermodynamic parameters of specific protein-protein, protein-DNA and small molecule interactions.
* 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
1. 1. Introduction 2. 2. Globular Protein Stability 3. 3. Mechanism and Limits of Amide 1H Exchange 4. 4. Requirements for Candidate Systems 5. 5. Preliminary Experiments 6. 6. A Protocol for Amide 1H Exchange 7. 7. Summary and Future Directions 8. Acknowledgments 9. Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion 10. Abbreviations 11. 1. Introduction: Co‐Translational Versus Post‐Translational Membrane Protein Insertion 12. 2. Challenges of Thermodynamic Analysis of Membrane Protein Folding/Insertion 13. 3. FCS and Protein–Membrane Interactions 14. 4. Thermodynamic Schemes for Analysis of Membrane Partitioning 15. 5. Kinetic Analysis of Membrane Protein Insertion 16. 6. Perspectives: Annexin B12 as a Model for Thermodynamic and Kinetic Analysis of Membrane Protein Insertion, Folding and Misfolding 17. Acknowledgments 18. Evaluating the Energy-Dependent “Binding” in the Early Stage of Protein Import into Chloroplasts 19. 1. Introduction 20. 2. The In Vitro Chloroplastic Import Assay Using Recombinant Precursor Proteins 21. 3. Limited Proteolysis of Docked Precursor Proteins 22. 4. The Behavior of Transit Peptide During the Transition 23. 5. Conclusions 24. Acknowledgments 25. Use of DNA Length Variation to Detect Periodicities in Positively Cooperative, Nonspecific Binding 26. 1. Introduction 27. 2. Protein and DNA Preparations 28. 3. Stoichiometry Analyses 29. 4. Affinity and Cooperativity as Functions of DNA Length 30. Acknowledgments 31. The Impact of Ions on Allosteric Functions in Human Liver Pyruvate Kinase 32. 1. Introduction 33. 2. General Strategy to Assess Allosteric Coupling 34. 3. PYK Assay 35. 4. Buffers 36. 5. Divalent Cation 37. 6. Monovalent Cation 38. 7. Anion 39. 8. Concluding Remarks 40. Acknowledgments 41. Conformational Stability of Cytochrome c Probed by Optical Spectroscopy 42. 1. Introduction 43. 2. Basic Theory of Absorption and Circular Dichroism Spectroscopy 44. 3. Secondary Structure Analysis of Cytochrome c Using Ultra-Violet Circular Dichroism Spectroscopy 45. 4. Visible CD and Absorption Spectroscopy of Native Cytochrome c 46. 5. Nonnative States of Ferricytochrome c Probed by Visible CD and Absorption Spectroscopy 47. 6. Summary and Outlook 48. Examining Ion Channel Properties Using Free-Energy Methods 49. 1. Introduction 50. 2. Free-Energy Calculations 51. 3. Thermodynamic Integration 52. 4. Free-Energy Perturbation 53. 5. Umbrella Sampling 54. 6. Adaptive Biasing Force 55. 7. Metadynamics 56. 8. Applications of Free-Energy Methods to Study Ion Channel Properties 57. 9. Conclusions and Future Outlook 58. Acknowledgments 59. Examining Cooperative Gating Phenomena in Voltage-Dependent Potassium Channels 60. 1. Introduction 61. 2. High-Order Thermodynamic Mutant Cycle Coupling Analysis 62. 3. The Voltage-Activated Potassium Channel Allosteric Model System 63. 4. Deriving a Hill Coefficient for Assessing Cooperativity in Voltage-Dependent Ion Channels 64. 5. Direct Analysis of Cooperativity in Multisubunit Allosteric Proteins 65. 6. Long-Range Energetic Coupling Mediated Through Allosteric Communication Trajectories 66. 7. Concluding Remarks 67. Acknowledgments 68. Thermal Stability of Collagen Triple Helix 69. 1. Introduction 70. 2. Methods 71. Electrostatic Contributions to the Stabilities of Native Proteins and Amyloid Complexes 72. 1. Introduction 73. 2. Practical Aspects of pKa Measurements by NMR 74. 3. Interpreting pKa Values in Terms of Stability 75. 4. Importance of the Reference (Unfolded) State 76. 5. Results from Globular Proteins 77. 6. Results from Coiled Coils 78. 7. Comparison of NMR and Crystallographic Results 79. 8. Comparison of NMR and Mutagenesis: Nonadditivity of Ion Pairs 80. 9. Improving Structure-Based Modeling of pKa Values 81. 10. Results with Micelle-Bound Proteins 82. 11. Results from Fibrillization Kinetics 83. 12. Conclusion 84. Acknowledgments 85. Kinetics of Allosteric Activation 86. 1. Linkage 87. 2. Allosteric Activation at Steady State 88. 3. Different Types of Activation (Type Ia, Type Ib, and Type II) 89. 4. Concluding Remarks 90. Acknowledgment 91. Thermodynamics of the Protein Translocation 92. 1. Introduction 93. 2. Example 1: SecA Nucleotide Binding 94. 3. Example 2: Probing SecB:Substrate Interactions 95. 4. Concluding Remarks 96. Thermodynamic Analysis of the Structure–Function Relationship in the Total DNA-Binding Site of Enzyme–DNA Complexes 97. Abbreviations 98. 1. Introduction 99. 2. Thermodynamic Bases of Quantitative Equilibrium Spectroscopic Titrations 100. 3. Anatomy of the Total DNA-Binding Site in the PriA Helicase–ssDNA Complex 101. 4. Structure–Function Relationship in the Total ssDNA-Binding Site of the DNA Repair Pol X From ASFV 102. Acknowledgments 103. Equilibrium and Kinetic Approaches for Studying Oligomeric Protein Folding 104. 1. Introduction 105. 2. Methods to Monitor Folding and Association 106. 3. Equilibrium Studies 107. 4. Kinetic Studies 108. Acknowledgments 109. Methods for Quantifying T cell Receptor Binding Affinities and Thermodynamics 110. 1. Introduction 111. 2. Isothermal Titration Calorimetry of TCR–Peptide/MHC Interactions 112. 3. Surface Plasmon Resonance Studies of TCR–Peptide/MHC Interactions 113. 4. Fluorescence Anisotropy as a Tool for Characterizing TCR–Peptide/MHC Interactions 114. 5. Concluding Remarks 115. Acknowledgments 116. Thermodynamic and Kinetic Analysis of Bromodomain–Histone Interactions 117. 1. Introduction 118. 2. Fluorescence Anisotropy Theory and Concepts 119. 3. Developing Binding Models for the Analysis of Fluorescence Anisotropy Data 120. 4. Experimental Considerations in Designing Fluorescence Anisotropy Assays 121. 5. Preparation of Histone and Bromodomain Samples 122. 6. Fluorescence Anisotropy Measurements 123. 7. Kinetic Analysis 124. 8. Determination of Thermodynamic Parameters 125. 9. Thermodynamic Measurements 126. 10. Developing a Binding Model 127. 11. Concluding Remarks 128. Acknowledgments 129. Chapter 17 Thermodynamics of 2-Cys Peroxiredoxin Assembly Determined by Isothermal Titration Calorimetry 130. Abbreviations 131. 1. Introduction 132. 2. Dimer–Decamer Equilibrium 133. 3. Isothermal Titration Calorimetry—General Concepts 134. 4. ITC Dilution Experiments 135. 5. Material and Instruments 136. 6. Experimental Procedure 137. 7. Results, Data Analysis, and Discussion 138. 8. Conclusions 139. Acknowledgments 140. Protein–Lipid Interactions 141. 1. Introduction 142. 2. Defining Protein–Lipid Interactions 143. 3. Selective Partitioning and Lipid Activities 144. 4. Protein–Protein Interactions at the Membrane Surface 145. 5. Measuring Protein–Lipid Interactions 146. 6. Modeling of Protein–Lipid Interactions 147. 7. Synopsis 148. Acknowledgments 149. Predicting pKa Values with Continuous Constant pH Molecular Dynamics 150. 1. Introduction 151. 2. Theoretical Methods for pKa Predictions 152. 3. Predicting Protein pKas with REX–CPHMD Simulations 153. 4. Conclusions 154. Acknowledgment 155. Unfolding Thermodynamics of DNA Intramolecular Complexes Involving Joined Triple- and Double-Helical Motifs 156. 1. Introduction 157. 2. Materials and Methods 158. 3. Results and Discussion 159. 4. Conclusions 160. Acknowledgments 161. Chapter 21 Thermodynamics and Conformational Change Governing Domain–Domain Interactions of Calmodulin 162. 1. Introduction 163. 2. Overexpression and Purification of rCaM Fragments 164. 3. Calcium-Binding Properties of N-Domain CaM Fragments 165. 4. Tertiary Constraints of N-Domain CaM Fragments 166. 5. Tertiary Conformation of N-Domain CaM Fragments 167. 6. High-Resolution Studies of N-Domain CaM Fragments 168. 7. Conclusions 169. Acknowledgments 170. Chapter 22 Use of Pressure Perturbation Calorimetry to Characterize the Volumetric Properties of Proteins 171. 1. Introduction 172. 2. Determination of the Coefficient of Thermal Expansion (αPr) Using PPC 173. 3. Sample Preparation 174. 4. Derivation of a Two-State Model for Analysis of PPC Data 175. 5. Practical Considerations 176. 6. Implications of Two-State Model for Future PPC Experiments 177. Chapter 23 Solvent Denaturation of Proteins and Interpretations of the m Value 178. 1. Introduction 179. 2. Protein Unfolding or Denaturation 180. 3. Linear Extrapolation Method 181. 4. ΔG(H2O): Conformational Stability 182. 5. The m Value 183. 6. Concluding Remarks 184. Acknowledgments 185. Chapter 24 Measuring Cotranslational Folding of Nascent Polypeptide Chains on Ribosomes 186. 1. Introduction 187. 2. Translation and the Ribosome:Nascent Chain (RNC) Complex 188. 3. General Approaches for Generating Stalled RNC Complexes 189. 4. Methods for Preparing RNC Complexes 190. 5. Biophysical Studies with RNC Complexes 191. 6. Measuring Nascent Chain Cotranslational Folding and Rigidity by Limited Protease Digestion 192. 7. Future Directions
The use of thermodynamics in biological research can be equated to 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. This volume presents sophisticated methods for estimating the thermodynamic parameters of specific protein-protein, protein-DNA and small molecule interactions.
* 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
1. 1. Introduction 2. 2. Globular Protein Stability 3. 3. Mechanism and Limits of Amide 1H Exchange 4. 4. Requirements for Candidate Systems 5. 5. Preliminary Experiments 6. 6. A Protocol for Amide 1H Exchange 7. 7. Summary and Future Directions 8. Acknowledgments 9. Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion 10. Abbreviations 11. 1. Introduction: Co‐Translational Versus Post‐Translational Membrane Protein Insertion 12. 2. Challenges of Thermodynamic Analysis of Membrane Protein Folding/Insertion 13. 3. FCS and Protein–Membrane Interactions 14. 4. Thermodynamic Schemes for Analysis of Membrane Partitioning 15. 5. Kinetic Analysis of Membrane Protein Insertion 16. 6. Perspectives: Annexin B12 as a Model for Thermodynamic and Kinetic Analysis of Membrane Protein Insertion, Folding and Misfolding 17. Acknowledgments 18. Evaluating the Energy-Dependent “Binding” in the Early Stage of Protein Import into Chloroplasts 19. 1. Introduction 20. 2. The In Vitro Chloroplastic Import Assay Using Recombinant Precursor Proteins 21. 3. Limited Proteolysis of Docked Precursor Proteins 22. 4. The Behavior of Transit Peptide During the Transition 23. 5. Conclusions 24. Acknowledgments 25. Use of DNA Length Variation to Detect Periodicities in Positively Cooperative, Nonspecific Binding 26. 1. Introduction 27. 2. Protein and DNA Preparations 28. 3. Stoichiometry Analyses 29. 4. Affinity and Cooperativity as Functions of DNA Length 30. Acknowledgments 31. The Impact of Ions on Allosteric Functions in Human Liver Pyruvate Kinase 32. 1. Introduction 33. 2. General Strategy to Assess Allosteric Coupling 34. 3. PYK Assay 35. 4. Buffers 36. 5. Divalent Cation 37. 6. Monovalent Cation 38. 7. Anion 39. 8. Concluding Remarks 40. Acknowledgments 41. Conformational Stability of Cytochrome c Probed by Optical Spectroscopy 42. 1. Introduction 43. 2. Basic Theory of Absorption and Circular Dichroism Spectroscopy 44. 3. Secondary Structure Analysis of Cytochrome c Using Ultra-Violet Circular Dichroism Spectroscopy 45. 4. Visible CD and Absorption Spectroscopy of Native Cytochrome c 46. 5. Nonnative States of Ferricytochrome c Probed by Visible CD and Absorption Spectroscopy 47. 6. Summary and Outlook 48. Examining Ion Channel Properties Using Free-Energy Methods 49. 1. Introduction 50. 2. Free-Energy Calculations 51. 3. Thermodynamic Integration 52. 4. Free-Energy Perturbation 53. 5. Umbrella Sampling 54. 6. Adaptive Biasing Force 55. 7. Metadynamics 56. 8. Applications of Free-Energy Methods to Study Ion Channel Properties 57. 9. Conclusions and Future Outlook 58. Acknowledgments 59. Examining Cooperative Gating Phenomena in Voltage-Dependent Potassium Channels 60. 1. Introduction 61. 2. High-Order Thermodynamic Mutant Cycle Coupling Analysis 62. 3. The Voltage-Activated Potassium Channel Allosteric Model System 63. 4. Deriving a Hill Coefficient for Assessing Cooperativity in Voltage-Dependent Ion Channels 64. 5. Direct Analysis of Cooperativity in Multisubunit Allosteric Proteins 65. 6. Long-Range Energetic Coupling Mediated Through Allosteric Communication Trajectories 66. 7. Concluding Remarks 67. Acknowledgments 68. Thermal Stability of Collagen Triple Helix 69. 1. Introduction 70. 2. Methods 71. Electrostatic Contributions to the Stabilities of Native Proteins and Amyloid Complexes 72. 1. Introduction 73. 2. Practical Aspects of pKa Measurements by NMR 74. 3. Interpreting pKa Values in Terms of Stability 75. 4. Importance of the Reference (Unfolded) State 76. 5. Results from Globular Proteins 77. 6. Results from Coiled Coils 78. 7. Comparison of NMR and Crystallographic Results 79. 8. Comparison of NMR and Mutagenesis: Nonadditivity of Ion Pairs 80. 9. Improving Structure-Based Modeling of pKa Values 81. 10. Results with Micelle-Bound Proteins 82. 11. Results from Fibrillization Kinetics 83. 12. Conclusion 84. Acknowledgments 85. Kinetics of Allosteric Activation 86. 1. Linkage 87. 2. Allosteric Activation at Steady State 88. 3. Different Types of Activation (Type Ia, Type Ib, and Type II) 89. 4. Concluding Remarks 90. Acknowledgment 91. Thermodynamics of the Protein Translocation 92. 1. Introduction 93. 2. Example 1: SecA Nucleotide Binding 94. 3. Example 2: Probing SecB:Substrate Interactions 95. 4. Concluding Remarks 96. Thermodynamic Analysis of the Structure–Function Relationship in the Total DNA-Binding Site of Enzyme–DNA Complexes 97. Abbreviations 98. 1. Introduction 99. 2. Thermodynamic Bases of Quantitative Equilibrium Spectroscopic Titrations 100. 3. Anatomy of the Total DNA-Binding Site in the PriA Helicase–ssDNA Complex 101. 4. Structure–Function Relationship in the Total ssDNA-Binding Site of the DNA Repair Pol X From ASFV 102. Acknowledgments 103. Equilibrium and Kinetic Approaches for Studying Oligomeric Protein Folding 104. 1. Introduction 105. 2. Methods to Monitor Folding and Association 106. 3. Equilibrium Studies 107. 4. Kinetic Studies 108. Acknowledgments 109. Methods for Quantifying T cell Receptor Binding Affinities and Thermodynamics 110. 1. Introduction 111. 2. Isothermal Titration Calorimetry of TCR–Peptide/MHC Interactions 112. 3. Surface Plasmon Resonance Studies of TCR–Peptide/MHC Interactions 113. 4. Fluorescence Anisotropy as a Tool for Characterizing TCR–Peptide/MHC Interactions 114. 5. Concluding Remarks 115. Acknowledgments 116. Thermodynamic and Kinetic Analysis of Bromodomain–Histone Interactions 117. 1. Introduction 118. 2. Fluorescence Anisotropy Theory and Concepts 119. 3. Developing Binding Models for the Analysis of Fluorescence Anisotropy Data 120. 4. Experimental Considerations in Designing Fluorescence Anisotropy Assays 121. 5. Preparation of Histone and Bromodomain Samples 122. 6. Fluorescence Anisotropy Measurements 123. 7. Kinetic Analysis 124. 8. Determination of Thermodynamic Parameters 125. 9. Thermodynamic Measurements 126. 10. Developing a Binding Model 127. 11. Concluding Remarks 128. Acknowledgments 129. Chapter 17 Thermodynamics of 2-Cys Peroxiredoxin Assembly Determined by Isothermal Titration Calorimetry 130. Abbreviations 131. 1. Introduction 132. 2. Dimer–Decamer Equilibrium 133. 3. Isothermal Titration Calorimetry—General Concepts 134. 4. ITC Dilution Experiments 135. 5. Material and Instruments 136. 6. Experimental Procedure 137. 7. Results, Data Analysis, and Discussion 138. 8. Conclusions 139. Acknowledgments 140. Protein–Lipid Interactions 141. 1. Introduction 142. 2. Defining Protein–Lipid Interactions 143. 3. Selective Partitioning and Lipid Activities 144. 4. Protein–Protein Interactions at the Membrane Surface 145. 5. Measuring Protein–Lipid Interactions 146. 6. Modeling of Protein–Lipid Interactions 147. 7. Synopsis 148. Acknowledgments 149. Predicting pKa Values with Continuous Constant pH Molecular Dynamics 150. 1. Introduction 151. 2. Theoretical Methods for pKa Predictions 152. 3. Predicting Protein pKas with REX–CPHMD Simulations 153. 4. Conclusions 154. Acknowledgment 155. Unfolding Thermodynamics of DNA Intramolecular Complexes Involving Joined Triple- and Double-Helical Motifs 156. 1. Introduction 157. 2. Materials and Methods 158. 3. Results and Discussion 159. 4. Conclusions 160. Acknowledgments 161. Chapter 21 Thermodynamics and Conformational Change Governing Domain–Domain Interactions of Calmodulin 162. 1. Introduction 163. 2. Overexpression and Purification of rCaM Fragments 164. 3. Calcium-Binding Properties of N-Domain CaM Fragments 165. 4. Tertiary Constraints of N-Domain CaM Fragments 166. 5. Tertiary Conformation of N-Domain CaM Fragments 167. 6. High-Resolution Studies of N-Domain CaM Fragments 168. 7. Conclusions 169. Acknowledgments 170. Chapter 22 Use of Pressure Perturbation Calorimetry to Characterize the Volumetric Properties of Proteins 171. 1. Introduction 172. 2. Determination of the Coefficient of Thermal Expansion (αPr) Using PPC 173. 3. Sample Preparation 174. 4. Derivation of a Two-State Model for Analysis of PPC Data 175. 5. Practical Considerations 176. 6. Implications of Two-State Model for Future PPC Experiments 177. Chapter 23 Solvent Denaturation of Proteins and Interpretations of the m Value 178. 1. Introduction 179. 2. Protein Unfolding or Denaturation 180. 3. Linear Extrapolation Method 181. 4. ΔG(H2O): Conformational Stability 182. 5. The m Value 183. 6. Concluding Remarks 184. Acknowledgments 185. Chapter 24 Measuring Cotranslational Folding of Nascent Polypeptide Chains on Ribosomes 186. 1. Introduction 187. 2. Translation and the Ribosome:Nascent Chain (RNC) Complex 188. 3. General Approaches for Generating Stalled RNC Complexes 189. 4. Methods for Preparing RNC Complexes 190. 5. Biophysical Studies with RNC Complexes 191. 6. Measuring Nascent Chain Cotranslational Folding and Rigidity by Limited Protease Digestion 192. 7. Future Directions
