Biothermodynamics, Part B

Using NMR-Detected Backbone Amide 1H Exchange to Assess Macromolecular Crowding Effects on Globular-Protein Stability
......Page 38
Introduction......Page 39
Mechanism and Limits of Amide 1H Exchange......Page 40
Requirements for Candidate Systems......Page 43
Polymer characterization......Page 45
Aggregation studies......Page 46
Protein-crowder interactions......Page 47
Exchange limit determination......Page 48
Intrinsic exchange rate......Page 50
A Protocol for Amide 1H Exchange......Page 51
Summary and Future Directions......Page 52
References......Page 53
Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion
......Page 56
Introduction: Co-Translational Versus Post-Translational Membrane Protein Insertion......Page 58
Experimental exploration of transmembrane insertion......Page 59
FCS experiment......Page 60
FCS data analysis......Page 61
Infinite dilution regime and FCS measurements at lipid saturation......Page 62
Lipid titration FCS measurements......Page 64
Thermodynamic Schemes for Analysis of Membrane Partitioning......Page 65
pH-dependent formation of membrane-competent state......Page 66
Interface-directed membrane insertion......Page 69
On the interpretation of quantitative kinetic analysis......Page 70
Spectroscopic measurements along W rarr I rarr T pathway
......Page 71
Example of kinetic analysis of membrane insertion of ANX......Page 72
Perspectives: Annexin B12 as a Model for Thermodynamic and Kinetic Analysis of Membrane Protein Insertion, Folding and Misfolding
......Page 75
References......Page 77
Evaluating the Energy-Dependent ``Binding´´ in the Early Stage of Protein Import into Chloroplasts
......Page 80
Introduction......Page 81
The In Vitro Chloroplastic Import Assay Using Recombinant Precursor Proteins
......Page 82
Preparations for recombinant precursor proteins
......Page 83
Modification of the precursor......Page 84
The docking and the import assay......Page 85
Docking under various energy conditions......Page 87
Limited Proteolysis of Docked Precursor Proteins......Page 90
Proteolytic fragments......Page 92
Transition between Stages II and III intermediates
......Page 94
The Behavior of Transit Peptide During the Transition
......Page 96
References......Page 99
Use of DNA Length Variation to Detect Periodicities in Positively Cooperative, Nonspecific Binding
......Page 102
Introduction......Page 103
Stoichiometry Analyses......Page 105
Affinity and Cooperativity as Functions of DNA Length
......Page 112
References......Page 115
The Impact of Ions on Allosteric Functions in Human Liver Pyruvate Kinase
......Page 119
Introduction......Page 120
General Strategy to Assess Allosteric Coupling......Page 122
PYK Assay......Page 124
Buffers......Page 127
Divalent Cation......Page 129
Monovalent Cation......Page 131
Anion......Page 136
Concluding Remarks......Page 139
References......Page 141
Conformational Stability of Cytochrome c Probed by Optical Spectroscopy
......Page 144
Introduction......Page 145
Basic Theory of Absorption and Circular Dichroism Spectroscopy
......Page 148
Secondary Structure Analysis of Cytochrome c Using Ultra-Violet Circular Dichroism Spectroscopy
......Page 152
Visible CD and Absorption Spectroscopy of Native Cytochrome c
......Page 156
Nonnative States of Ferricytochrome c Probed by Visible CD and Absorption Spectroscopy
......Page 166
Summary and Outlook......Page 183
References......Page 184
Examining Ion Channel Properties Using Free-Energy Methods
......Page 189
Introduction......Page 190
Free-Energy Calculations......Page 191
Thermodynamic Integration......Page 193
Free-Energy Perturbation......Page 194
Umbrella Sampling......Page 196
Adaptive Biasing Force......Page 198
Metadynamics......Page 201
Free-energy perturbation and ion permeation and selectivity
......Page 203
Umbrella sampling and conduction mechanisms......Page 204
Metadynamics and gating at the selectivity filter of K+ channels
......Page 206
Conclusions and Future Outlook......Page 208
References......Page 209
Examining Cooperative Gating Phenomena in Voltage-Dependent Potassium Channels: Taking the Energetic Approach
......Page 212
Introduction......Page 213
High-Order Thermodynamic Mutant Cycle Coupling Analysis
......Page 214
Double-mutant cycle coupling analysis......Page 215
High-order mutant cycle coupling analysis......Page 218
The Voltage-Activated Potassium Channel Allosteric Model System
......Page 221
The analogy between voltage-dependent ion channels and allosteric enzymes
......Page 226
Understanding the meaning of the Hill coefficient
......Page 227
Direct Analysis of Cooperativity in Multisubunit Allosteric Proteins
......Page 229
The high-order coupling pattern of intersubunit interactions as a criterion for discerning concerted or sequential conformational transitions
......Page 230
Direct demonstration for concerted pore opening in Kv channels
......Page 232
Long-Range Energetic Coupling Mediated Through Allosteric Communication Trajectories
......Page 235
High-order coupling analysis of Kv channel allosteric communication trajectories: Emerging principles
......Page 237
References......Page 240
Thermal Stability of Collagen Triple Helix
......Page 243
Introduction......Page 244
Characterization of the thermostability of protein
......Page 246
The thermal denaturation of collagen
......Page 248
Characterization of the subdomains-The conclusions of the DSC study of collagen
......Page 252
The reversible, two-state thermal transition of triple helical peptides
......Page 253
The propensity scale for triple helix
......Page 257
The microunfolding of a subdomain of the collagen is modulated by the long-range impacts of the local interactions
......Page 259
References......Page 263
Electrostatic Contributions to the Stabilities of Native Proteins and Amyloid Complexes
......Page 265
Introduction......Page 266
Practical Aspects of pKa Measurements by NMR
......Page 268
Results from Globular Proteins......Page 272
Results from Coiled Coils......Page 273
Comparison of NMR and Crystallographic Results
......Page 274
Comparison of NMR and Mutagenesis: Nonadditivity of Ion Pairs
......Page 275
Improving Structure-Based Modeling of pKa Values
......Page 276
Results with Micelle-Bound Proteins......Page 277
Results from Fibrillization Kinetics......Page 281
Conclusion......Page 285
References......Page 286
Linkage......Page 291
Allosteric Activation at Steady State......Page 293
Different Types of Activation (Type Ia, Type Ib, and Type II)
......Page 298
Concluding Remarks......Page 301
References......Page 302
Thermodynamics of the Protein Translocation
......Page 304
Protein translocation......Page 305
Isothermal titration calorimetry......Page 307
Data analysis......Page 308
Example 1: SecA Nucleotide Binding......Page 309
Materials and methods......Page 310
Experimental part......Page 311
Temperature dependence......Page 312
Example 2: Probing SecB:Substrate Interactions
......Page 314
Materials and methods......Page 315
Experimental part......Page 316
Concluding Remarks......Page 319
References......Page 320
Thermodynamic Analysis of the Structure-Function Relationship in the Total DNA-Binding Site of Enzyme-DNA Complexes
......Page 323
Introduction......Page 324
Thermodynamic Bases of Quantitative Equilibrium Spectroscopic Titrations
......Page 326
The site-size of the ssDNA-binding site proper of the PriA-ssDNA complex
......Page 332
Macroscopic affinities of PriA-ssDNA complexes containing a single PriA protein molecule bound
......Page 337
Model of PriA protein-ssDNA interactions
......Page 340
Intrinsic affinities of PriA-ssDNA interactions
......Page 343
Binding modes in PriA-ssDNA interactions
......Page 346
Model of the ASFV Pol X-ssDNA interactions
......Page 347
Model of the ASFV Pol X-ssDNA complex
......Page 350
References......Page 352
Equilibrium and Kinetic Approaches for Studying Oligomeric Protein Folding......Page 355
Introduction......Page 356
Perturbation methods......Page 357
Circular dichroism......Page 360
Fluorescence......Page 361
Förster resonance energy transfer......Page 362
Monitoring radius of gyration and molecular weight......Page 365
General approach......Page 366
Specific considerations for data collection......Page 367
Data analysis for two-state unfolding......Page 370
Identifying equilibrium intermediates......Page 372
General approach......Page 373
Data analysis......Page 375
Burst-phase reactions......Page 376
Analyzing kinetic traces......Page 380
Transient kinetic intermediates......Page 382
References......Page 384
Methods for Quantifying T cell Receptor Binding Affinities and Thermodynamics......Page 388
Introduction......Page 389
Concentration requirements and data quality......Page 391
Linkage effects in calorimetric experiments......Page 393
Other practical concerns for titration calorimetry......Page 395
Introduction to SPR......Page 396
Use of SPR to measure low-affinity TCR-peptide/MHC interactions......Page 397
Underlying binding thermodynamics from SPR experiments......Page 401
Introduction to fluorescence anisotropy......Page 402
An example TCR-peptide/MHC interaction characterized by fluorescence anisotropy......Page 404
References......Page 407
Thermodynamic and Kinetic Analysis of Bromodomain-Histone Interactions......Page 411
Fluorescence Anisotropy Theory and Concepts......Page 412
Developing Binding Models for the Analysis of Fluorescence Anisotropy Data......Page 414
Experimental Considerations in Designing Fluorescence Anisotropy Assays......Page 418
Preparation of Histone and Bromodomain Samples......Page 419
Fluorescence Anisotropy Measurements......Page 420
Kinetic Analysis......Page 423
Determination of Thermodynamic Parameters......Page 427
Thermodynamic Measurements......Page 428
Developing a Binding Model......Page 431
References......Page 433
Thermodynamics of 2-Cys Peroxiredoxin Assembly Determined by Isothermal Titration Calorimetry......Page 436
Introduction......Page 437
Dimer-Decamer Equilibrium......Page 439
Isothermal Titration Calorimetry-General Concepts......Page 442
ITC Dilution Experiments......Page 443
2-CysPrx purification......Page 445
Selection of Prx concentration in the syringe......Page 446
Determination of protein concentration......Page 447
Equilibration period......Page 448
Loading the syringe into the cell......Page 449
Protocol one......Page 450
Protocol two......Page 454
References......Page 455
Protein-Lipid Interactions: Role of Membrane Plasticity and Lipid Specificity on Peripheral Protein Interactions......Page 458
Introduction......Page 459
Defining Protein-Lipid Interactions......Page 460
Selective Partitioning and Lipid Activities......Page 461
Protein-Protein Interactions at the Membrane Surface......Page 462
The general use of a signal: Lever rule derivation......Page 464
Extension beyond fluorescence spectroscopy......Page 465
Interpretation of binding partition functions......Page 466
Comparison to Langmuir model......Page 467
Binding partition functions: Linked binding equilibrium......Page 468
Isothermal titration calorimetry to measure protein-lipid interactions......Page 470
Modeling protein-protein interactions......Page 472
Synopsis......Page 475
Acknowledgments......Page 477
References......Page 478
Predicting pKa Values with Continuous Constant pH Molecular Dynamics......Page 481
Introduction......Page 482
Theoretical Methods for pKa Predictions......Page 483
Methods based on macroscopic description of proteins......Page 484
Methods based on discrete protonation states......Page 486
Methods based on continuous protonation states......Page 487
Addressing issues in the microscopic methods......Page 489
Surface residues in RNase A......Page 491
Buried groups in staphylococcal nuclease......Page 493
Conformational fluctuations in α-lactalbumin......Page 495
Conclusions......Page 496
References......Page 497
Unfolding Thermodynamics of DNA Intramolecular Complexes Involving Joined Triple- and Double-Helical Motifs......Page 502
Introduction......Page 503
Temperature-dependent UV spectroscopy (UV melts)......Page 506
Differential scanning calorimetry......Page 508
Differential binding of protons and counterions......Page 509
Overall experimental protocol......Page 510
UV unfolding of complexes......Page 511
CD spectroscopy and complex conformation......Page 512
Calorimetric unfolding of DNA complexes......Page 513
Melting behavior of complexes relative to its control triplex and hairpin loops......Page 517
Differential binding of protons and counterions in the folding of complexes......Page 519
Unfolding of complexes: Enthalpy contributions of their triplex and duplex motifs......Page 522
Acknowledgments......Page 524
References......Page 525
Thermodynamics and Conformational Change Governing Domain-Domain Interactions of Calmodulin
......Page 528
Introduction......Page 529
Experimental conditions......Page 532
Analysis of free energy of calcium binding......Page 534
Fluorimeter and cuvettes......Page 535
Ca2+-indicator dye......Page 536
Experimental conditions......Page 537
Analysis of thermal denaturation......Page 539
Buffer selection......Page 540
Tertiary Conformation of N-Domain CaM Fragments......Page 541
Experimental conditions......Page 542
Ca2+-independent behavior of calibration standards......Page 543
High-Resolution Studies of N-Domain CaM Fragments......Page 544
Optimization of crystallization conditions......Page 545
Diffraction data collection and processing......Page 546
Conclusions......Page 547
Acknowledgments......Page 549
References......Page 550
Use of Pressure Perturbation Calorimetry to Characterize the Volumetric Properties of Proteins
......Page 552
Introduction......Page 553
Determination of the Coefficient of Thermal Expansion (alphaPr) Using PPC
......Page 556
Sample Preparation......Page 558
Derivation of a Two-State Model for Analysis of PPC Data......Page 560
Absolute values of alpha for native and unfolded proteins and their temperature dependencies
......Page 564
Importance of DeltaH for quantitative estimates of DeltaV/V......Page 566
Example of global analysis of PPC data and P-T diagram......Page 568
References......Page 570
Introduction......Page 573
Protein Unfolding or Denaturation......Page 574
Linear Extrapolation Method......Page 579
DeltaG(H2O): Conformational Stability......Page 580
The m Value......Page 582
Concluding Remarks......Page 586
References......Page 587
Measuring Cotranslational Folding of Nascent Polypeptide Chains on Ribosomes......Page 590
Introduction......Page 591
Translation and the Ribosome:Nascent Chain (RNC) Complex......Page 593
General Approaches for Generating Stalled RNC Complexes......Page 595
Nascent chain sequence-based production of RNC complexes......Page 596
Methods for Preparing RNC Complexes......Page 600
Procedure......Page 601
Biophysical Studies with RNC Complexes......Page 602
Cotranslational folding studies......Page 603
Studies of nascent chain dynamics......Page 604
Complementary approaches to cotranslational folding......Page 605
Preliminary considerations......Page 606
Future Directions......Page 607
References......Page 608