Biothermodynamics, Part B (eBook)

Front Cover 1
Methods in Enzymology 4
Copyright Page 5
Contents 6
Contributors 14
Preface 20
Chapter 1: Using NMR-Detected Backbone Amide 1H Exchange to assess Macromolecular Crowding Effects on Globular-Protein Stability 50
1. Introduction 51
2. Globular Protein Stability 52
3. Mechanism and Limits of Amide 1H Exchange 52
4. Requirements for Candidate Systems 55
5. Preliminary Experiments 57
6. A Protocol for Amide 1H Exchange 63
7. Summary and Future Directions 64
Acknowledgments 65
References 65
Chapter 2: Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion 68
1. Introduction: Co-Translational Versus Post-Translational Membrane Protein Insertion 70
2. Challenges of Thermodynamic Analysis of Membrane Protein Folding/Insertion 71
3. FCS and Protein-Membrane Interactions 72
4. Thermodynamic Schemes for Analysis of Membrane Partitioning 77
5. Kinetic Analysis of Membrane Protein Insertion 81
6 Perspectives: Annexin B12 as a Model for Thermodynamic and Kinetic Analysis of Membrane Protein Insertion, Folding and Misfolding 87
Acknowledgments 89
References 89
Chapter 3: Evaluating the Energy-Dependent ``Binding´´ in the Early Stage of Protein Import into Chloroplasts 92
1. Introduction 93
2. The In Vitro Chloroplastic Import Assay Using Recombinant Precursor Proteins 94
3. Limited Proteolysis of Docked Precursor Proteins 102
4. The Behavior of Transit Peptide During the Transition 108
5. Conclusions 111
Acknowledgments 111
References 111
Chapter 4: Use of DNA Length Variation to Detect Periodicities in Positively Cooperative, Nonspecific Binding 114
1. Introduction 115
2. Protein and DNA Preparations 117
3. Stoichiometry Analyses 117
4. Affinity and Cooperativity as Functions of DNA Length 124
Acknowledgments 127
References 127
Chapter 5: The Impact of Ions on Allosteric Functions in Human Liver Pyruvate Kinase 132
1. Introduction 133
2. General Strategy to Assess Allosteric Coupling 135
3. PYK Assay 137
4. Buffers 140
5. Divalent Cation 142
6. Monovalent Cation 144
7. Anion 149
8. Concluding Remarks 152
Acknowledgments 154
References 154
Chapter 6: Conformational Stability of Cytochrome c Probed by Optical Spectroscopy 158
1. Introduction 159
2. Basic Theory of Absorption and Circular Dichroism Spectroscopy 162
3. Secondary Structure Analysis of Cytochrome c Using Ultra-Violet Circular Dichroism Spectroscopy 166
4. Visible CD and Absorption Spectroscopy of Native Cytochrome c 170
5. Nonnative States of Ferricytochrome c Probed by Visible CD and Absorption Spectroscopy 180
6. Summary and Outlook 197
References 198
Chapter 7: Examining Ion Channel Properties Using Free-Energy Methods 204
1. Introduction 205
2. Free-Energy Calculations 206
3. Thermodynamic Integration 208
4. Free-Energy Perturbation 209
5. Umbrella Sampling 211
6. Adaptive Biasing Force 213
7. Metadynamics 216
8. Applications of Free-Energy Methods to Study Ion Channel Properties 218
9. Conclusions and Future Outlook 223
Acknowledgments 224
References 224
Chapter 8: Examining Cooperative Gating Phenomena in Voltage-Dependent Potassium Channels: Taking the Energetic Approach 228
1. Introduction 229
2. High-Order Thermodynamic Mutant Cycle Coupling Analysis 230
3. The Voltage-Activated Potassium Channel Allosteric Model System 237
4. Deriving a Hill Coefficient for Assessing Cooperativity in Voltage-Dependent Ion Channels 242
5. Direct Analysis of Cooperativity in Multisubunit Allosteric Proteins 245
6. Long-Range Energetic Coupling Mediated Through Allosteric Communication Trajectories 251
7. Concluding Remarks 256
Acknowledgments 256
References 256
Chapter 9: Thermal Stability of Collagen Triple Helix 260
1. Introduction 261
2. Methods 263
References 280
Chapter 10: Electrostatic Contributions to the Stabilities of Native Proteins and Amyloid Complexes 282
1. Introduction 283
2. Practical Aspects of pKa Measurements by NMR 285
3. Interpreting pKa Values in Terms of Stability 289
4. Importance of the Reference (Unfolded) State 289
5. Results from Globular Proteins 289
6. Results from Coiled Coils 290
7. Comparison of NMR and Crystallographic Results 291
8. Comparison of NMR and Mutagenesis: Nonadditivity of Ion Pairs 292
9. Improving Structure-Based Modeling of pKa Values 293
10. Results with Micelle-Bound Proteins 294
11. Results from Fibrillization Kinetics 298
12. Conclusion 302
Acknowledgments 303
References 303
Chapter 11: Kinetics of Allosteric Activation 308
1. Linkage 308
2. Allosteric Activation at Steady State 310
3. Different Types of Activation (Type Ia, Type Ib, and Type II) 315
4. Concluding Remarks 318
Acknowledgment 319
References 319
Chapter 12: Thermodynamics of the Protein Translocation 322
1. Introduction 323
2. Example 1: SecA Nucleotide Binding 327
3. Example 2: Probing SecB:Substrate Interactions 332
4. Concluding Remarks 337
References 338
Chapter 13: Thermodynamic Analysis of the Structure-Function Relationship in the Total DNA-Binding Site of Enzyme-DNA Complexes 342
1. Introduction 343
2. Thermodynamic Bases of Quantitative Equilibrium Spectroscopic Titrations 345
3. Anatomy of the Total DNA-Binding Site in the PriA Helicase-ssDNA Complex 351
4. Structure-Function Relationship in the Total ssDNA-Binding Site of the DNA Repair Pol X From ASFV 366
Acknowledgments 371
References 371
Chapter 14: Equilibrium and Kinetic Approaches for Studying Oligomeric Protein Folding 374
1. Introduction 375
2. Methods to Monitor Folding and Association 376
3. Equilibrium Studies 385
4. Kinetic Studies 392
Acknowledgments 403
References 403
Chapter 15: Methods for Quantifying T cell Receptor Binding Affinities and Thermodynamics 408
1. Introduction 409
2. Isothermal Titration Calorimetry of TCR-Peptide/MHC Interactions 411
3. Surface Plasmon Resonance Studies of TCR-Peptide/MHC Interactions 416
4. Fluorescence Anisotropy as a Tool for Characterizing TCR-Peptide/MHC Interactions 422
5. Concluding Remarks 427
Acknowledgments 427
References 427
Chapter 16: Thermodynamic and Kinetic Analysis of Bromodomain-Histone Interactions 432
1. Introduction 433
2. Fluorescence Anisotropy Theory and Concepts 433
3. Developing Binding Models for the Analysis of Fluorescence Anisotropy Data 435
4. Experimental Considerations in Designing Fluorescence Anisotropy Assays 439
5. Preparation of Histone and Bromodomain Samples 440
6. Fluorescence Anisotropy Measurements 441
7. Kinetic Analysis 444
8. Determination of Thermodynamic Parameters 448
9. Thermodynamic Measurements 449
10. Developing a Binding Model 452
11. Concluding Remarks 454
Acknowledgments 454
References 454
Chapter 17: Thermodynamics of 2-Cys Peroxiredoxin Assembly Determined by Isothermal Titration Calorimetry 458
1. Introduction 459
2. Dimer-Decamer Equilibrium 461
3. Isothermal Titration Calorimetry-General Concepts 464
4. ITC Dilution Experiments 465
5. Material and Instruments 467
6. Experimental Procedure 467
7. Results, Data Analysis, and Discussion 472
8. Conclusions 477
Acknowledgments 477
References 477
Chapter 18: Protein-Lipid Interactions: Role of Membrane Plasticity and Lipid Specificity on Peripheral Protein Interactions 480
1. Introduction 481
2. Defining Protein-Lipid Interactions 482
3. Selective Partitioning and Lipid Activities 483
4. Protein-Protein Interactions at the Membrane Surface 484
5. Measuring Protein-Lipid Interactions 486
6. Modeling of Protein-Lipid Interactions 488
7. Synopsis 497
Acknowledgments 499
References 500
Chapter 19: Predicting pKa Values with Continuous Constant pH Molecular Dynamics 504
1. Introduction 505
2. Theoretical Methods for pKa Predictions 506
3. Predicting Protein pKas with REX-CPHMD Simulations 514
4. Conclusions 519
Acknowledgment 520
References 520
Chapter 20: Unfolding Thermodynamics of DNA Intramolecular Complexes Involving Joined Triple- and Double-Helical Motifs 526
1. Introduction 527
2. Materials and Methods 530
3. Results and Discussion 534
4. Conclusions 548
Acknowledgments 548
References 549
Chapter 21: Thermodynamics and Conformational Change Governing Domain-Domain Interactions of Calmodulin 552
1. Introduction 553
2. Overexpression and Purification of rCaM Fragments 556
3. Calcium-Binding Properties of N-Domain CaM Fragments 556
4. Tertiary Constraints of N-Domain CaM Fragments 561
5. Tertiary Conformation of N-Domain CaM Fragments 565
6. High-Resolution Studies of N-Domain CaM Fragments 568
7. Conclusions 571
Acknowledgments 573
References 574
Chapter 22: Use of Pressure Perturbation Calorimetry to Characterize the Volumetric Properties of Proteins 576
1. Introduction 577
2. Determination of the Coefficient of Thermal Expansion (aPr) Using PPC 580
3. Sample Preparation 582
4. Derivation of a Two-State Model for Analysis of PPC Data 584
5. Practical Considerations 588
6. Implications of Two-State Model for Future PPC Experiments 594
References 594
Chapter 23: Solvent Denaturation of Proteins and Interpretations of the m Value 598
1. Introduction 598
2. Protein Unfolding or Denaturation 599
3. Linear Extrapolation Method 604
4. DeltaG(H2O): Conformational Stability 605
5. The m Value 607
6. Concluding Remarks 611
Acknowledgments 612
References 612
Chapter 24: Measuring Cotranslational Folding of Nascent Polypeptide Chains on Ribosomes 616
1. Introduction 617
2. Translation and the Ribosome:Nascent Chain (RNC) Complex 619
3. General Approaches for Generating Stalled RNC Complexes 621
4. Methods for Preparing RNC Complexes 626
5. Biophysical Studies with RNC Complexes 628
6. Measuring Nascent Chain Cotranslational Folding and Rigidity by Limited Protease Digestion 632
7. Future Directions 633
References 634
Author Index 640
Subject Index 652
Color Plates 664