Protein NMR Techniques (Methods in Molecular Biology, v831)

Cover......Page 1
Frontmatter......Page 4
Preface......Page 6
Contents......Page 10
Contributors......Page 12
1. Introduction......Page 16
2.1. Sample Preparation and SDS-PAGE......Page 17
2.2. Protein Expression......Page 18
2.3. Protein Purification......Page 19
3.1. PAGE Sample Preparation......Page 20
3.2. SDS-PAGE......Page 21
3.3. A Proper Starting Culture......Page 22
3.5. Double Colony Selection......Page 23
3.6. High Cell-Density IPTG-Induction Method......Page 25
3.7. Optimization of Various Conditions......Page 26
3.9. Conclusion......Page 29
4. Notes......Page 30
References......Page 33
1. Introduction......Page 34
1.1. The P. pastoris Expression System......Page 35
1.2. The K. lactis Expression System......Page 36
2.1. Uniform 13 C, 15 N-Labeling Using P. pastoris......Page 37
2.2. Uniform 2 H, 15 N-Labeling Using P. pastoris......Page 38
2.3. Uniform 13 C, 15 N-Labeling Using K. lactis......Page 39
3.1. Uniform 13 C, 15 N-Labeling Using P. pastoris......Page 41
3.2. Uniform 2 H, 15 N-Labeling Using P. pastoris......Page 43
3.4. Uniform 2 H, 15 N-Labeling Using K. lactis......Page 45
4. Notes......Page 47
References......Page 50
1. Introduction: Baculovirus-Insect Cell Expression System......Page 52
1.1. Principle of Baculovirus-Mediated Recombinant Protein Expression......Page 55
1.2. Commercially Available Baculovirus Expression Systems......Page 56
1.4. Expression of Labeled Recombinant Protein in the Baculovirus Expression System......Page 58
2.1. Cotransfection of Insect Cells......Page 59
3. Methods......Page 60
3.1. Cotransfection of Insect Cells......Page 61
3.3. Analysis of Recombinant Protein Expression......Page 62
3.4. Production of Isotopically Labeled Protein......Page 64
4. Notes......Page 65
References......Page 67
1.1. Principle of Mammalian Cell-Mediated Protein Expression......Page 70
1.2. Mammalian Cell Lines......Page 72
1.3. Expression and Labeling of Recombinant Proteins in Mammalian Cells......Page 73
2.2. Specific Isotope Labeling of Proteins......Page 76
3.1. Expression of Isotope-Labeled Recombinant Proteins......Page 78
3.2. Specific Isotope Labeling of Proteins......Page 80
4. Conclusions......Page 81
5. Notes......Page 82
References......Page 83
1. Introduction......Page 86
2.1. Preparation of E. coli S30 Extract......Page 88
2.3. Cell-Free Synthesis Reaction......Page 89
3. Methods......Page 90
3.1. Preparation of E. coli S30 Extract......Page 92
3.2. Preparation of T7 RNA Polymerase......Page 94
3.3. Cell-Free Synthesis Reaction......Page 96
4. Notes......Page 97
References......Page 99
1. Introduction......Page 100
2.1. Cell-Free Expression of the Mechanosensitive Channel MscL in Detergent Micelles......Page 102
2.2. Cell-Free Expression of the Mechanosensitive Channel MscL in Liposomes......Page 105
2.3. Sample Characterization......Page 107
2.4. NMR Sample Preparation......Page 108
3.1.1. Day 1: Cell-Free Protein Expression in Detergent Micelles......Page 109
3.1.2. Day 2: Purification of Detergent/Protein Micelles......Page 110
3.1.3. Day 3: Sample Characterization and Reconstitution into Liposomes......Page 111
3.1.4. Day 4: Preparation of the Sample for Solid-State NMR......Page 112
3.2.1. Day 1: Cell-Free Protein Expression......Page 113
3.2.2. Day 2: Preparation of the Sample for Solid-State NMR......Page 114
3.3.3. Electrophoresis for Oligomeric State Characterization......Page 115
3.3.4. Dot Blot......Page 116
3.3.5. Semidry Western-Blot......Page 117
3.3.7. Protein Quantification in Proteoliposomes......Page 118
3.3.9. Proteoliposome Density Characterization......Page 119
3.4. NMR Sample Preparation......Page 120
4. Notes......Page 121
References......Page 123
1. Introduction......Page 126
2. Expression of the Catalytic Domain of c-Src in Escherichia coli......Page 130
3.2. Media for Bacterial Growth......Page 133
3.3. Protein Purification......Page 135
4.1.1. Overexpression of SrcCD......Page 136
4.1.2. Protocol for the Purification of SrcCD from the MBP Fusion Protein......Page 138
4.2. Expression and Purification of the Fully Active, Wild-Type Catalytic Domain of c-Src......Page 139
5. Conclusions......Page 140
6. Notes......Page 141
References......Page 144
1. Introduction......Page 148
3.1. Protein Labeling for Methyl Detection......Page 150
3.3. Paramagnetic Relaxation Enhancement Measurements......Page 152
4. Notes......Page 153
References......Page 155
1. Introduction......Page 156
1.1. Theory......Page 157
3.1. Preliminary Set-up......Page 159
3.2.1. Longitudinal Relaxation Rate......Page 161
3.2.2. Transverse Relaxation Rates with a Single Echo......Page 164
3.3. 15 N–{1H} Nuclear Overhauser Effect Measurements\r......Page 166
3.4. Measurements of CSA/DD Cross-Correlated Cross-Relaxation Rates......Page 168
3.4.2. Longitudinal Cross-Correlated Cross-Relaxation Rates......Page 170
3.5. Analysis and Interpretation of 15 N Relaxation Rates......Page 171
3.5.2. A Note on the Model-Free Analysis of 15 N Relaxation Rates......Page 172
4. Notes......Page 173
References......Page 176
1. Introduction......Page 180
2.2. Fusion Protein Purification, Cleavage, and Preparation of Pure M2......Page 183
2.5. Structure Calculation and Measurement of Tryptophan Gate Dynamics......Page 184
3. Methods......Page 185
3.2. Fusion Protein Purification, Cleavage, and Preparation of Pure M2......Page 186
3.4. NMR Chemical Shift Assignments and Restraint Measurements......Page 187
3.5. Structure Calculation and Measurement of Tryptophan Gate Dynamics......Page 190
4. Notes......Page 191
References......Page 193
1. Introduction......Page 196
2.1. Cell Transformation......Page 197
2.3.2. Minimal Medium......Page 198
2.6.2. Ni-NTA Chromatography......Page 199
3. Methods......Page 200
3.2. Cell Expression Testing......Page 201
3.3.1. LB Medium......Page 202
3.4.2. Minimal Medium and Deuterated Minimal Medium......Page 203
3.6.1. Cell Lysis......Page 204
3.6.3. Desalting Exchange and Heparin Chromatography......Page 205
3.6.4. Superdex 200 Gel Filtration Chromatography......Page 206
4. Notes......Page 207
References......Page 209
1. Introduction......Page 212
2.2. RNA Synthesis......Page 214
2.5. RNA–Protein Interactions by NMR......Page 215
3.1. DNA Template Design for RNA Synthesis......Page 216
3.2. RNA Synthesis......Page 217
3.3. RNA Purification......Page 220
3.4. Preliminary RNA Analysis by NMR......Page 223
3.5. RNA–Protein Interactions by NMR......Page 225
4. Notes......Page 227
References......Page 232
1. Introduction......Page 234
2.1. Binding Affinity Measurements......Page 237
2.4. Preparation of the Protein–DNA Complex......Page 238
3.1. Binding Affinity Measurements......Page 239
3.2. Preparation of Purified Single-Stranded DNA......Page 240
3.4. Preparation of the Protein–DNA Complex......Page 241
3.5. Assessing the Quality of the Protein–DNA Complex......Page 243
4. Notes......Page 244
References......Page 246
1. Introduction......Page 248
1.1. Protein Observed Experiments......Page 250
1.2. Ligand Observed Experiments......Page 254
2.1. Protein-Based Study on the Binding of VEGF to the Peptidic Ligand P-7i......Page 260
3.1.1. Sample Preparation......Page 261
3.1.2. Recording of CSP NMR Spectra......Page 262
3.1.3. Data Analysis......Page 263
3.2. Ligand-Based Study on Binding \rof POP to the Ligand Baicalin......Page 265
3.2.1. Optimization of STD Parameters and Confirmation of Baicalin as a POP Ligand......Page 266
3.2.2. Identifying the Binding Epitope on Baicalin......Page 268
4. Notes......Page 269
References......Page 272
1.1. In-Cell NMR Spectroscopy: The Technique and Information It Reveals......Page 276
1.2. In-Cell NMR Spectroscopy: Current Achievements and Challenges......Page 277
1.3. In-Cell NMR Spectroscopy: Looking Ahead......Page 282
2.1. Protein Expression......Page 283
2.3. Freeze–Thaw Lysis......Page 284
3.1. Protein Expression for Preparing an In-Cell NMR Sample......Page 285
3.2. Cell Viability Assay ( 41) ( see Note 3)......Page 287
4. Notes......Page 288
References......Page 290
1. Introduction......Page 294
2. Correlation Spectroscopy......Page 296
3. Characterization of Dynamics in the Solid State......Page 302
References......Page 309
1. Introduction......Page 318
2.1. Preparation of Thioredoxin Reassemblies for Solid-State NMR Studies......Page 325
2.2. Preparation of HIV-1 CA Assemblies......Page 327
2.3. Transmission Electron Microscopy of HIV-1 CA Protein Assemblies......Page 328
2.6. Preparation of CAP-Gly/Microtubule Complexes......Page 329
3.1. Preparation of Thioredoxin Reassemblies for Solid-State NMR Studies......Page 330
3.2. Preparation of HIV-1 CA Assemblies......Page 331
3.3. Transmission Electron Microscopy of HIV-1 CA Protein Assemblies and CAP-Gly/Microtubule Protein Assemblies......Page 332
3.5. Cryo-SEM Microscopy of HIV-1 CA Protein Assemblies......Page 333
3.6. Preparation of CAP-Gly/Microtubule Complexes......Page 334
3.8. Solid-State NMR Spectroscopy for Resonance Assignments......Page 335
3.9. Solid-State NMR Experiments for Probing Protein Backbone Dynamics......Page 336
3.10. Solid-State NMR Experiments for Structural Analysis of Protein Interfaces......Page 338
4. Notes......Page 340
References......Page 343
1. Introduction......Page 348
1.1.1. Solid-Phase Peptide Synthesis......Page 349
1.1.2. Protein Overexpression......Page 350
1.2.1. Peptide Purification......Page 351
1.2.2. Protein Purification......Page 352
1.3.1. Mass Spectrometry......Page 353
1.3.3. Polarized Attenuated Total Reflection Fourier Transform Infrared Spectroscopy......Page 354
1.3.4. Circular Dichroism Spectroscopy......Page 355
1.4.1. Solid-State 2 H NMR Spectroscopy......Page 356
1.4.2. Solution-State NMR Spectroscopy......Page 357
2. Materials......Page 358
2.2.2. Protein Purification, Organic and Aqueous Extractions......Page 359
2.4.1. Reconstitution into Membrane Lipids......Page 360
3.2.1. Peptide Purification......Page 361
3.2.2. Protein Purification......Page 362
3.2.5. Detergent Exchange......Page 363
3.4.1. Reconstitution into Membrane Lipids......Page 364
3.4.2. Solubilization in Detergent Micelles......Page 365
4. Notes......Page 366
References......Page 369
1. Introduction......Page 374
2.1. Standard Triple-Resonance Experiments......Page 375
2.2. Use of Predicted Chemical Shifts......Page 376
2.3. Use of Structural Information......Page 377
2.4. Use of Selective Labeling Strategies......Page 378
2.5. Use of Spin-Labeled ATP Analogs......Page 380
3. Conclusions......Page 381
References......Page 382
1. Introduction......Page 384
2.1. Hydrogen Exchange as a Measure of Solvent Accessibility......Page 385
2.2. Comparing Solvent Accessibility from Molecular Simulations to that Inferred from the Steric Interpretation of Hydrogen Exchange......Page 388
3.1. Implications of Amides Being Weak Normal Eigen Acids......Page 390
3.2. Electronic Polarizability in the Dielectric Shielding of the Peptide Anion......Page 391
4.1. Magnetization Transfer Methods......Page 393
4.2. Solvent Exchange by 1 H Exchange-In Protocol......Page 394
5.1. Electrostatic Parameter Set Dependence of Peptide Acidity Predictions......Page 395
5.2. Atomic Charge Distribution in the Peptide Anion......Page 397
5.3. Dominant Acidic Conformer Analysis......Page 399
6.1. Population Averaging of Conformer Acidities......Page 400
6.2. Consistency of Ubiquitin Model Ensembles with the Native State Conformational Distribution......Page 401
6.3. Comparison Against the Set of Known Ubiquitin-Protein Complexes......Page 403
6.4. Hydrogen Exchange Analysis as a Monitor for Completeness of Ensemble Sampling......Page 405
7.1. Backbone Conformation Dependence of Side Chain Correction Factors for Hydrogen Exchange......Page 406
7.2. Dependence of Peptide Acidity on Backbone Conformation......Page 407
7.3. Hydrogen Exchange for Nonpolar Side Chains and Nonadditivity of Correction Factors......Page 410
7.4. Evidence for Dielectric Shielding from Conformational Reorganization in Carboxamide Side Chains......Page 412
7.5. Deviations in Dielectric Continuum Modeling of Hydrogen Exchange Arising from Chemical Induction and Aspartate Side Chain Interactions......Page 413
References......Page 415
1. Introduction......Page 422
2. Description of the Various Tools Implemented in BATCH......Page 423
2.2. ASCOM 15 N Spectral Width for Rapid Sampling of the 15 N Dimension......Page 424
2.3. COBRA for Automated Extraction of Sequential Connectivities......Page 425
2.5. The HADAMAC Experiment for Amino Acid Typing......Page 426
2.6. 13 C Chemical Shift Extraction......Page 427
3.2. NMR Hardware and Pulse Sequences......Page 428
4.2. The 15 N HSQC Experiment......Page 429
4.3. The HADAMAC Experiment......Page 430
4.4. The Triple Resonance Experiments......Page 431
5.1. General Parameters......Page 432
5.2. Preprocessing of the Raw Data......Page 434
6.1. 2D Peak List Generation and Extraction of Amino Acid Type......Page 435
6.2. COBRA Analysis......Page 436
7. Backbone Assignment......Page 439
8. 1 H, 15 N, and 13 C Chemical Shift Extraction......Page 440
9. Conclusions......Page 441
References......Page 442
1. Introduction......Page 444
2. Specificities of the UNIO Program......Page 447
3. Standard UNIO Protocol for Protein NMR Structure Determination......Page 448
4.1. Overview of the ATNOS Algorithm......Page 450
5.1. Overview of the MATCH Algorithm......Page 453
5.3. APSY-NMR Input Data......Page 455
6.1. Overview of the ATNOS/ASCAN Approach......Page 456
7.1. Overview of the ATNOS/CANDID Approach......Page 458
7.2. Ambiguous Distance Restraints......Page 461
7.3. Network-Anchored NOE Assignment......Page 462
References......Page 463
1. Introduction......Page 468
2.2. Input Data......Page 470
3.1.1. Data Filtering......Page 471
3.2.1. Chemical-Shift Based Assignment......Page 472
3.2.2. Structural Rules for Symmetric Oligomers......Page 473
3.3.1. Ambiguous Distance Restraints......Page 474
3.3.2. Distance Calibration......Page 475
3.3.4. Partial Assignment......Page 476
3.3.5. Calculation of Structure Ensemble......Page 477
3.3.8. Floating Chirality Assignment......Page 479
3.5.1. Export to CCPN......Page 480
3.6. Conversion of Input Data......Page 481
3.7. Specification of ARIA Project Parameters......Page 483
3.9. Starting an ARIA Run......Page 485
3.10.1. Convergence......Page 486
3.10.2. Automated Assignments......Page 487
3.10.3. Quality Indices......Page 488
3.11.2. Adjusting Parameters......Page 490
4. Notes......Page 491
References......Page 495
1. Introduction......Page 500
1.1. The Underlying Equations......Page 501
2.1. Highlights of the Program DYNAMICS......Page 504
2.2. The Overall Organization of the Program......Page 505
2.3. Treating the Overall Rotational Diffusion of a Molecule......Page 506
2.4. Selection of the Appropriate Model for Local Motion......Page 507
2.5.3. Run-Time Dialog......Page 509
2.5.4. Understanding the Output......Page 517
2.6. Practical Examples......Page 519
3.2. Strategy for Determining the Overall Rotational Correlation Time When the Structure is Unknown or RotDif Results are Unreliable......Page 520
3.6. Auxiliary Programs......Page 523
3.6.4. Demo Scripts......Page 524
References......Page 525
Index\r......Page 528