Biothermodynamics, Part 3

Front matter......Page 0
Copyright......Page 2
Contributors......Page 3
Preface......Page 6
Methods In Enzymology......Page 7
Measurement and Analysis of Equilibrium Binding Titrations......Page 36
Monitoring a Binding Reaction......Page 37
The Binding Equation and Its Relationship to Binding Measurements......Page 40
Plotting and Analysis of Binding Data......Page 43
Protein Concentration Is Important: Equilibrium Versus Stoichiometric Conditions......Page 45
Deviations from Simple Binding......Page 49
References......Page 51
Macromolecular Competition Titration Method......Page 52
Introduction......Page 53
A Single Titration Curve: Some Simple Considerations of Possible Pitfalls......Page 55
Quantitative Equilibrium Spectroscopic Titrations: Thermodynamic Bases......Page 59
Nucleotide Binding to the RepA Protein of Plasmid RSF1010......Page 64
Applying the Statistical Thermodynamic Model for the Nucleotide Binding to the RSF1010 RepA Protein Hexamer.........Page 67
Empirical Function Approach......Page 70
MCT Method: General Considerations......Page 71
Application of the MCT Method to the Base Specificity Problem in ASFV Pol X-ssDNA System......Page 74
Application of MCT Method to Protein-ssDNA Lattice Binding Systems......Page 76
Quantitative Analysis of the Binding of the E. coli DnaB Helicase to Unmodified Nucleic Acids Using the MCT Method.........Page 81
Direct Analysis of the Experimental Isotherm of Protein Ligand Binding to Two Competing Nucleic Acid Lattices.........Page 83
Using a Single Concentration of a Nonfluorescent Unmodified Nucleic Acid......Page 87
Using Short Fluorescent Oligonucleotides in Competition with the Polymer Nucleic Acid......Page 88
Acknowledgments......Page 90
References......Page 91
Analysis of PKR-RNA Interactions by Sedimentation Velocity......Page 93
Introduction......Page 94
Reagents and Cells......Page 96
Experimental Design......Page 98
Data analysis......Page 99
20bp dsRNA......Page 101
TAR RNA dimer......Page 104
VAI......Page 107
Acknowledgments......Page 109
References......Page 110
Structural and Thermodynamic Analysis of PDZ-Ligand Interactions......Page 114
Introduction......Page 115
Structural Studies of the Tiam1 PDZ Domain......Page 116
Theoretical background......Page 120
Experimental design considerations......Page 123
Synthetic peptides......Page 124
Equilibrium fluorescence binding assays......Page 125
Double-Mutant Cycle Analysis of PDZ-Binding Pockets......Page 126
The peptide evolution strategy......Page 128
Double-mutant cycle analysis of evolved peptides......Page 129
Conclusions......Page 131
References......Page 132
Thermodynamic Analysis of Metal Ion-Induced Protein Assembly......Page 134
Introduction......Page 135
Linked Equilibria-General Concepts......Page 136
Sedimentation velocity......Page 139
Sedimentation equilibrium......Page 144
Other experimental approaches......Page 149
Summary......Page 151
References......Page 152
Thermodynamic Dissection of Colicin Interactions......Page 155
Introduction......Page 156
Measuring equilibrium dissociation constants......Page 159
Sample considerations......Page 160
Baseline corrections......Page 161
Data presentation......Page 162
DNase Domain-Immunity Protein Interactions......Page 164
The impact of zinc on DNase-Im thermodynamics......Page 165
Thermodynamics of noncognate DNase-Im complexes......Page 166
Measuring subnanomolar Kds through competitive ITC......Page 168
Interaction of colicin N (ColN) with trimeric porins......Page 169
Formation of the BtuB-colicin E9 complex......Page 170
Interaction of TolB with Pal......Page 172
Mapping the TolB binding site within colicin E9......Page 174
Discussion......Page 175
References......Page 176
Energetics of Src Homology Domain Interactions in Receptor Tyrosine Kinase-Mediated Signaling......Page 178
Introduction......Page 179
Interactions of Src Homology 2 Domains......Page 180
Recognition by the ``Two-Pinned Plug´´......Page 184
Recognition by the beta-Turn Motif......Page 190
Selectivity Versus Specificity for SH2 Domain Interactions......Page 191
Proline Sequence-Recognition Domains......Page 193
Interactions of SH3 Domains......Page 194
What Constitutes Specificity in SH3 Domain Interactions?......Page 200
Selectivity in SH3 Domain Interactions......Page 202
Interactions Through Multiple Domains......Page 205
Conclusions......Page 207
References......Page 208
Structural and Functional Energetic Linkages in Allosteric Regulation of Muscle Pyruvate Kinase......Page 215
Wyman linked function......Page 217
Reinhart derivation of the Weber expression as applied to steady-state kinetics......Page 218
Functional Energetic Linkages in Allosteric Regulation of Rabbit Muscle Pyruvate Kinase......Page 219
Functional Linkage Through Steady-State Kinetics......Page 220
Coupling reaction between H+ and Phe......Page 221
Coupling reactions between metal ions and other metabolites......Page 222
Structural Perturbations by Ligands......Page 223
Differential sedimentation velocity......Page 224
Analytical gel filtration chromatography......Page 226
The SANS data can be subjected to two separate analyses......Page 229
Comparison of solution and crystal structures......Page 230
Functional Linkage Scheme of Allostery for RMPK......Page 231
Functional Linkage Through Ligand Binding Measurements......Page 232
Equilibrium binding......Page 233
Isothermal titration calorimetry......Page 234
Global fitting......Page 235
PEP binding......Page 236
Proton release or absorption......Page 237
Protein Structural Dynamics-Amide Hydrogen Exchange Monitored by FT-IR (HX-FT-IR)......Page 239
S402P mutation in the interface between the C-domains......Page 240
T340M mutation along the interface between the A-domains......Page 241
Summary Statement......Page 242
References......Page 243
Analysis of Free Energy Versus Temperature Curves in Protein Folding and Macromolecular Interactions......Page 248
Stability Curves=Gibbs-Helmholtz Curves=DeltaG Versus Temperature......Page 249
Analysis of DeltaG Versus Temperature in Protein Folding......Page 252
Using Stability Curves to Compare Mesophilic and Thermophilic Protein Pairs......Page 254
Temperature Dependence of Folding Enthalpies and Entropies......Page 255
Analysis of DeltaG Versus Temperature Data in Macromolecular Interactions......Page 258
Fitting DeltaH and DeltaG Versus Temperature for a DeltaDeltaCp......Page 259
Examples of Potential Consequences of a Small DeltaDeltaCp......Page 264
References......Page 266
Application of the Sequential n-Step Kinetic Mechanism to Polypeptide Translocases......Page 268
Introduction......Page 269
Single-Turnover Fluorescence Stopped-Flow Method to Monitor Polypeptide Translocation......Page 270
Substrate design......Page 272
Enzyme trap......Page 274
Application of the Sequential n-Step Mechanism......Page 275
n-step mechanism with equivalent rate-constants......Page 276
Biphasic kinetics......Page 279
Finite processivity......Page 282
Determination of kinetic step-size......Page 285
ClpA catalyzed polypeptide translocation......Page 290
Concluding Remarks......Page 291
References......Page 292
Abstract......Page 294
Introduction......Page 295
Salt-Mediated Nucleosome (Dis)Assembly......Page 296
A Chaperone-Mediated Coupled Approach to Nucleosome Thermodynamics......Page 298
Fluorescence titrations......Page 304
Simple binary affinity measurements......Page 305
Chaperone-based assay to measure nucleosome thermodynamics (coupled equilibrium assay)......Page 306
Hill coefficients......Page 310
Summary and Implications......Page 311
References......Page 313
Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo......Page 315
Introduction......Page 316
Worm-like chain (WLC) model......Page 317
The j-factor......Page 319
DNA cyclization kinetics theory......Page 320
Representative protocols for DNA probe design, preparation, labeling, and quantitation......Page 322
Purification of HMGB proteins......Page 324
DNA ligation kinetics......Page 325
Data fitting and ODE approach......Page 326
Example data......Page 329
Concept......Page 330
Experimental design......Page 331
Data handling and curve fitting......Page 335
Reporter constructs for DNA looping in bacteria......Page 336
Disruption of hupA and hupB genes......Page 337
Protein expression......Page 338
E. coli beta-galactosidase reporter assays......Page 339
Example data and analysis......Page 340
R Code for j-Factor Experiments1......Page 342
R Code for lac Looping Experiments......Page 347
Required Files for R scripts......Page 360
References......Page 361
B......Page 364
D......Page 365
G......Page 366
H......Page 367
K......Page 368
M......Page 369
O......Page 370
R......Page 371
S......Page 372
V......Page 373
Z......Page 374
E......Page 375
L......Page 376
N......Page 377
R......Page 378
S......Page 379
W......Page 380