Biothermodynamics, Part D

Series Editor......Page 1
Copyright......Page 2
Contributors......Page 3
Preface......Page 7
Methods in Enzymology......Page 9
A Thermodynamic Approach for the Targeting of Nucleic Acid Structures Using Their Complementary Single Strands......Page 38
Introduction......Page 39
Materials......Page 41
Isothermal titration calorimetry......Page 42
Overall experimental approach......Page 43
Targeting an intramolecular G-quadruplex, G2......Page 45
Targeting an intramolecular triplex of the pyrimidine motif......Page 48
Targeting stem-loop motifs......Page 50
Targeting a DNA pseudoknot......Page 54
Targeting of three-way junctions......Page 57
Thermodynamic profiles for the targeting reactions......Page 59
Conclusions......Page 60
References......Page 61
Thermodynamics of Biological Processes......Page 64
Introduction: Thermodynamics is Not Just for Dead Stuff......Page 65
States and Weights from the Boltzmann Rule......Page 66
Thermodynamic models of binding......Page 70
Thermodynamic models of transcription......Page 73
MWC and hemoglobin: Where it all began......Page 77
MWC and ligand-gated ion channels: Cooperative gating......Page 79
MWC and chemotaxis: Cooperativity in signal detection......Page 81
MWC and eukaryotic transcriptional regulation: From nucleosomes to enhancers......Page 86
The biological reach of MWC models......Page 88
The Unreasonable Effectiveness of Random-Walk Models......Page 89
Conclusions......Page 92
References......Page 93
Protein Stability in the Presence of Cosolutes......Page 97
Introduction......Page 98
Protein stability......Page 99
General outline of how to measure isothermal protein unfolding......Page 100
Case 1: Adenylate kinase unfolding by urea......Page 108
Curve fitting: Basics......Page 110
Curve fitting: Looking beyond the obvious......Page 111
General outline how to use osmolyte mixtures......Page 114
Case 2: Nank isothermal folding/unfolding in urea-sarcosine mixtures......Page 116
Data treatment with mixed osmolytes: Independent analysis of the individual curves......Page 117
Global analysis......Page 121
General outline......Page 122
Case 3: von Willebrand factor: two-state unfolding of the collagen-binding A3 domain......Page 124
Data treatment: Independent analysis of the individual curves......Page 125
Global analysis......Page 127
General outline how to use osmolytes in thermal scans......Page 128
Data treatment: Independent analysis of the individual curves......Page 132
Global analysis......Page 135
What is a Taylor expansion?......Page 137
Standard Taylor expansion......Page 139
Classical protein stability curve: A nonstandard Taylor expansion......Page 141
Issues with the classical stability equation......Page 143
A better approach: Standard Taylor expansion......Page 144
Implementing parameters that are already known......Page 147
Protein stability as a function of temperature and the concentrations of two osmolytes......Page 148
Phase diagrams......Page 151
Three-state versus two-state transitions......Page 153
Oligomeric versus monomeric proteins......Page 154
References......Page 157
Small-Angle X-ray Scattering Studies of Peptide-Lipid Interactions Using the Mouse Paneth Cell α-Defensin Cryptdin-4\0......Page 162
Cell-penetrating peptides......Page 163
Antimicrobial peptides......Page 164
α-Defensins\0......Page 165
Peptide-induced membrane restructuring......Page 166
X-Rays as Structural Probes of Biological Systems Under Biomimetic Conditions......Page 167
X-ray diffraction of weakly ordered systems......Page 168
Theory of X-ray diffraction......Page 169
Preparation of recombinant α-defensins\0......Page 172
Purification of recombinant α-defensins\0......Page 173
Refolding of recombinant and synthetic peptides......Page 174
Microbicidal peptide assays......Page 175
Data collection......Page 176
Translation of two-dimensional X-ray image to diffraction data......Page 177
References......Page 180
Synergy of Molecular Dynamics and Isothermal Titration Calorimetry in Studies of Allostery......Page 185
Allostery......Page 186
Arginine Repressor......Page 188
Preparation for Simulations......Page 191
Sampling of States......Page 193
Equilibration......Page 194
Correlated Motions......Page 197
Structural Features of Correlated Motions......Page 198
Arg Residues Promote Rotation and Oscillation......Page 201
Single-Arginine Simulations......Page 203
Rotational Ensembles......Page 204
Energetic Contributions......Page 207
Reconciliation with Crystallographic Data......Page 211
Complementarity and Synergy of MD and ITC......Page 213
Prospects......Page 218
References......Page 220
Using Tryptophan Fluorescence to Measure the Stability of Membrane Proteins Folded in Liposomes......Page 223
Introduction......Page 224
The contribution of light scattering to a tryptophan fluorescence emission scan can be divested from true tryptophan emis......Page 225
Effects of light scattering on the tryptophan fluorescence from membrane proteins......Page 227
Reducing light scattering by refractive index matching......Page 229
Rayleigh-Gans-Debye theory describes light scattering by liposomes......Page 231
Position-width analysis......Page 234
Variation of tryptophan spectral properties with fractional populations of folded protein......Page 236
Choosing an Appropriate Tryptophan Spectral Property to Measure the Thermodynamic Stabilities of Folded Membrane Proteins........Page 239
Conclusions......Page 241
Protein folding reactions......Page 243
References......Page 244
Non-B Conformations of CAG Repeats Using 2-Aminopurine......Page 246
Diseases associated with repeated sequences......Page 247
2-Aminopurine as a structural and energetic probe......Page 248
Conformational integrity......Page 249
Calculation of thermodynamic parameters......Page 251
Oligonucleotide concentration......Page 252
Acrylamide quenching......Page 255
(CAG)8 hairpin......Page 256
(CAG)8 three-way junction......Page 260
References......Page 262
Disulfide Bond-Mediated Passenger Domain Stalling as a Structural Probe of Autotransporter Outer Membrane Secretion in Vivo......Page 265
The Autotransporter Secretion Pathway......Page 266
Architecture and Processing of AT Passenger Domains......Page 268
Heterologous Passenger Domain Secretion......Page 271
Selecting a Model Autotransporter for Cys-Loop Stalling......Page 272
Disulfide bond formation in the periplasm......Page 273
Designing cysteine pairs for Cys-loop stalling......Page 274
beta-Mercaptoethanol-dependent stalling......Page 275
Preliminary considerations......Page 276
Preliminary conditions......Page 279
Applications: Using Cys-Loop Stalling to Define the Mechanism of AT OM Secretion......Page 280
References......Page 281
Strategies for the Thermodynamic Characterization of Linked Binding/Local Folding Reactions Within the Native Stat.........Page 284
Introduction......Page 285
A Mutation Strategy to Amplify Locally Unfolded States......Page 287
Thermodynamic Properties of Linked Folding and Binding Reactions......Page 291
Strategies for Quantitative Interpretation of Measured Enthalpies for a Linked Folding and Binding System......Page 296
Interplay of Local Mutational Effects, Global Stability, and Binding Affinity......Page 299
Success of the Strategy in Preserving Structure......Page 304
Comparison of Interaction Versus Entropy Based Mutation Strategy......Page 305
How Similar Are Local and Global Unfolding?......Page 307
Summary......Page 310
References......Page 311
Fluorescence-Detected Sedimentation in Dilute and Highly Concentrated Solutions......Page 314
Overview of AUC......Page 315
Fluorescence Optics for the Ultracentrifuge......Page 316
Sensitivity......Page 318
Labeling......Page 319
Characterization of products......Page 320
Degree of labeling......Page 321
Site specificity of conjugation......Page 322
Dye-protein interaction......Page 323
Applications of AU-FDS......Page 324
NUTS......Page 325
BOLTS......Page 326
BOLTS for pathophysiologic studies......Page 328
Signal nonlinearity and accuracy......Page 329
Dye-protein interaction in NUTS......Page 331
Conclusion......Page 332
References......Page 333
Author Index......Page 336
Subject Index......Page 347