Biothermodynamics

Cover Page......Page 1
Title Page......Page 2
Copyright Page......Page 3
Contributors......Page 4
Preface......Page 7
Practical Approaches to Protein Folding and Assembly: Spectroscopic Strategies in Thermodynamics and Kinetics......Page 35
Introduction......Page 36
Practical considerations......Page 37
Fluorescence emission......Page 39
Preparation of 10 M urea stock......Page 40
Confirm that the protein is completely unfolded......Page 42
Method 1......Page 43
Equilibrium unfolding......Page 44
Interpretation of equilibrium-unfolding curves......Page 46
Monomeric models......Page 49
Dimeric Models......Page 50
Equilibrium constants and fractions of species......Page 53
General considerations......Page 55
Sample preparation for measuring refolding and unfolding kinetics......Page 57
Instrument procedure......Page 58
Differential quenching by acrylamide......Page 60
Experimental procedure......Page 61
Burst phase......Page 63
Exponential fits......Page 64
Simulations......Page 66
References......Page 70
Using Thermodynamics to Understand Progesterone Receptor Function: Method and Theory......Page 74
Introduction......Page 75
Assessing Protein Functional and Structural Homogeneity......Page 76
Dissecting Linked Assembly Reactions......Page 79
Analysis and Dissection of Natural Promoters......Page 87
Measuring the Energetics of Coactivator Recruitment......Page 95
Correlation to Biological Function......Page 97
Conclusions and Future Directions......Page 100
References......Page 101
Direct Quantitation of Mg2+-RNA Interactions by Use of a Fluorescent Dye......Page 104
Introduction......Page 105
Ion-RNA interactions described by preferential interaction coefficients......Page 106
Using an Mg2+-binding dye to measure Gamma2+......Page 107
Interaction coefficients vs. binding densities......Page 109
Ion-Binding Properties of HQS......Page 111
Reagents and stock solutions......Page 114
Sample preparation......Page 116
Automated titrations......Page 117
Manual titrations......Page 120
Data Analysis......Page 121
Controls and Further Considerations......Page 123
References......Page 125
Analysis of Repeat-Protein Folding Using Nearest-Neighbor Statistical Mechanical Models......Page 128
Application to linear biopolymers......Page 129
Linear Repeat Proteins and Their Connection to Linear Ising Models......Page 130
Formulating a Homopolymer Partition Function and the Zipper Approximation......Page 133
Matrix Approach: Homopolymers......Page 137
Matrix Approach: Heteropolymers......Page 142
Solvability Criteria for Ising Models Applied to Repeat-Protein Folding......Page 144
Matrix Homopolymer Analysis of Consensus TPR Folding......Page 148
Matrix Heteropolymer Analysis of Consensus Ankyrin Repeat Folding......Page 152
Summary and Future Directions......Page 156
References......Page 157
Isothermal Titration Calorimetry: General Formalism Using Binding Polynomials......Page 159
Introduction......Page 160
The Binding Polynomial......Page 161
Microscopic Constants and Cooperativity......Page 163
Independent or Cooperative Binding?......Page 164
Analysis of ITC Data Using Binding Polynomials......Page 165
A Typical Case: Macromolecule with Two Ligand-Binding Sites......Page 167
Data Analysis......Page 169
Data Interpretation......Page 173
Independent ligand binding: Two identical binding sites......Page 174
Independent ligand binding: Two nonidentical binding sites......Page 175
Cooperative ligand binding: Two identical binding sites......Page 176
An Experimental Example......Page 178
Experimental Situations from the Literature......Page 179
Conclusions......Page 182
Appendix......Page 183
References......Page 186
Kinetic and Equilibrium Analysis of the Myosin ATPase......Page 188
Introduction......Page 189
Reagents and Equipment Used for all Assays......Page 190
High salt ATPase activity of myosin......Page 192
Actin-activated Mg2+-ATPase activity of myosin......Page 193
Sedimentation assays......Page 197
Pyrene fluorescence measurements......Page 199
Transient Kinetic Analysis of the Individual ATPase Cycle Transitions......Page 201
Myosin binding to and dissociation from actin......Page 202
Method......Page 204
ATP binding and hydrolysis by myosin......Page 206
Intrinsic tryptophan fluorescence......Page 207
Quench flow......Page 208
Actin-activated Pi release......Page 210
ATP binding to an equilibrated mixture of actomyosin and ADP......Page 213
ATP and ADP binding to actomyosin......Page 216
Kinetic Simulations......Page 219
Acknowledgments......Page 220
References......Page 221
The Hill Coefficient: Inadequate Resolution of Cooperativity in Human Hemoglobin......Page 224
Cooperativity and Intrinsic Binding......Page 225
The Macroscopic Binding Isotherm......Page 228
The Hill Coefficient......Page 231
Formulation of the Adair constants......Page 234
Redefinition of the Hill coefficient by Wyman......Page 235
Microscopic Cooperativity in Hemoglobin......Page 236
The hemoglobin binding cascade......Page 237
Insensitivity of the binding isotherm......Page 240
Summary......Page 242
References......Page 243
Methods for Measuring the Thermodynamic Stability of Membrane Proteins......Page 244
Introduction......Page 245
Two Classes of Membrane Proteins......Page 246
Methods for Measuring Transmembrane Domain Oligomer Stability......Page 247
Fooumlrster resonance energy transfer (FRET)......Page 248
Genetic assay systems (TOXCAT, POSSYCAT, and GALLEX)......Page 249
Methods for Measuring Multipass alpha-helical Membrane Protein Stability......Page 250
Methods to Study the Stability of beta-barrel Membrane Proteins......Page 253
Thermal denaturation......Page 254
Solvent denaturation with urea or GdnHCl......Page 255
Van der Waals/packing interactions......Page 258
Aromatic-aromatic interactions......Page 259
Elastic lipid bilayer forces......Page 260
Conclusion and Outlook......Page 262
References......Page 263
NMR Analysis of Dynein Light Chain Dimerization and Interactions with Diverse Ligands......Page 268
NMR Methodology......Page 269
Monomer-dimer Equilibrium Coupled to Electrostatics......Page 272
Dimerization is Coupled to Ligand Binding......Page 277
Folding is Coupled to Binding......Page 278
Allostery in LC8......Page 282
Summary......Page 286
References......Page 287
Characterization of Parvalbumin and Polcalcin Divalent Ion Binding by Isothermal Titration Calorimetry......Page 290
Introduction......Page 291
Practical Aspects of Data Collection......Page 293
Standardization of metal ion and chelator solutions......Page 294
Preparation of EDTA-agarose......Page 295
Removal of Ca2+ from protein samples......Page 296
Binding parameters for competing chelators......Page 298
Data set preparation......Page 299
General comments on ITC model development......Page 302
Binding in the presence of a competing chelator......Page 306
Binding in the presence of a competing metal ion......Page 307
Least-squares minimization......Page 308
Error analysis......Page 311
The independent two-site model......Page 312
Competing chelator......Page 314
Competing metal ion......Page 315
Analysis of the divalent ion binding by the S55D/E59D variant of rat alpha-parvalbumin......Page 316
Analysis of positively cooperative divalent ion binding......Page 319
Modeling divalent ion binding by Phl p 7......Page 322
References......Page 326
Energetic Profiling of Protein Folds......Page 329
Introduction......Page 330
Modeling the Native State Ensemble of Proteins using Statistical Thermodynamics......Page 331
Energetic Profiles of Proteins Derived from Thermodynamics of the Native State Ensemble......Page 334
Principal Components Analysis of Energetic Profile Space......Page 336
Energetic Profiles are Conserved Between Homologous Proteins......Page 338
Direct Alignment of Energetic Profiles Based on a Variant of the CE Algorithm......Page 345
CE Algorithm Described for Structure Coordinates......Page 346
Necessary Deviations from the CE Algorithm to Accommodate Energetic Profiles......Page 347
Towards a Thermodynamic Homology of Fold Space: Clustering Energetic Profiles using STEPH......Page 348
Energetic Profiles Provide a Vehicle to Discover Conserved Substructures in the Absence of Known Homology......Page 351
Conclusion......Page 353
References......Page 355
Model Membrane Thermodynamics and Lateral Distribution of Cholesterol: From Experimental Data to Monte Carlo Simulation......Page 358
Introduction......Page 359
Liposome preparation for COD activity measurement......Page 360
Liposome preparation for X-ray diffraction measurement......Page 361
Cholesterol oxidase activity assay......Page 362
Monte Carlo simulation of lipid membranes using a lattice model......Page 363
Pairwise interactions and multibody interactions......Page 364
Calculation of chemical potential of cholesterol from simulation......Page 365
Maximum solubility of cholesterol in PC bilayers......Page 367
Multibody interactions and jumps in cholesterol chemical potential......Page 370
Physical origin of the cholesterol multibody interaction: The umbrella model......Page 375
The intrinsic connection between a regular distribution and a jump in chemical potential......Page 378
The competition between cholesterol and ceramide in POPC bilayers......Page 379
Current conceptual models of cholesterol-lipid interactions......Page 381
Chemical potential of cholesterol in DOPC, POPC, and DPPC bilayers explored by COD activity assay......Page 384
Comparison of models of cholesterol-lipid interaction......Page 388
Quantitative indication of cholesterol affinity to lipid bilayers......Page 390
Acknowledgments......Page 391
References......Page 392
Thinking Inside the Box: Designing, Implementing, and Interpreting Thermodynamic Cycles to Dissect Cooperativity in RNA and DNA Folding......Page 394
Introduction......Page 395
Folding Cooperativity Defined......Page 396
Thermodynamic Boxes: Design, Implementation, and Interpretation......Page 398
Thermodynamic Cubes: Design, Implementation, and Interpretation......Page 403
Examples of Cooperativity in RNA......Page 405
Secondary structure......Page 406
Tertiary structure......Page 407
Measuring Thermodynamic Parameters by UV Melting......Page 408
Sample preparation......Page 409
Extinction coefficient......Page 410
Concentration independence......Page 411
Buffer choice......Page 412
Running an experiment......Page 413
Melt fit equations......Page 414
Nonlinear curve fitting with KaleidaGraph......Page 418
Concluding Remarks......Page 419
References......Page 420
The Thermodynamics of Virus Capsid Assembly......Page 423
Introduction......Page 424
Virus capsid geometry......Page 425
Interaction specificity as a means of assembly regulation......Page 427
Subunit interactions......Page 428
Macromolecular polymerization and classical nucleation theory......Page 429
Thermodynamic theory of capsid assembly......Page 431
Methods of analysis......Page 433
Applications of Thermodynamic Evaluation of Virus Capsid Stability......Page 436
Cowpea chlorotic mottle virus......Page 437
Hepatitis B virus......Page 438
Bacteriophages......Page 440
References......Page 442
Extracting Equilibrium Constants from Kinetically Limited Reacting Systems......Page 446
Introduction......Page 447
Simulation and Analysis of Dimerization......Page 448
Kinetically Mediated Dimerization......Page 455
A Stepwise Approach......Page 463
Final Thoughts......Page 469
References......Page 470
Author Index......Page 474
Subject Index......Page 487