Membrane Protein Structure Determination: Methods and Protocols (Methods in Molecular Biology, Vol. 654)

Cover......Page 1
Methods in Molecular Biology, Vol. 654......Page 2
Membrane Protein StructureDetermination......Page 4
ISBN 9781607617617......Page 5
Preface......Page 6
Contents......Page 8
Contributors......Page 10
Part I:
Membrane Protein Purification......Page 15
1. Introduction......Page 17
2. Materials......Page 19
3.1. Detergent Concentration Determination......Page 20
3.2. Detergent Removal Using Bio-Beads......Page 23
3.3. Proteoliposomes Formation......Page 25
3.4. Ligand Binding......Page 27
4. Notes......Page 30
References......Page 31
1. Introduction......Page 33
2.2. Buffer Compositions......Page 34
3.2. The 9-Histidine Tag: Mutagenesis, Plasmids, and Strains......Page 35
3.5. AAH6 Purification......Page 36
3.7. Protein Concentration......Page 37
3.9. Purification of bAncp......Page 38
3.10.1. Practical Aspects......Page 39
3.10.2. The Genetic Approach......Page 40
References......Page 41
1. Introduction......Page 43
2.1. Expression......Page 44
2.2. Purification......Page 45
2.3. Characterization......Page 46
3.2.2. Large-Scale Production in a 5-L Bioreactor......Page 47
3.2.3. Production of Labelled Protein for NMR Studies......Page 48
3.3.1. Solubilization of Inclusion Bodies......Page 49
3.3.3. Small-Scale Purification, Manual Chromatographic Procedure......Page 50
3.3.4. General Considerations on IMAC Purification......Page 52
3.4.1. Protein Composition and Oligomeric State......Page 54
3.4.2. Optimal Detergent Concentration......Page 55
4. Conclusions......Page 56
5. Notes......Page 57
Acknowledgment......Page 58
References......Page 59
1. Introduction......Page 61
2.2. Molecular Biology......Page 63
2.3. Biochemistry......Page 64
3.1.1.2. Obtaining Mutant Strains by Host Selection......Page 67
3.1.1.3. ABCG2 Overexpression Using a Selected Strain......Page 69
3.1.2.1. Insect Cell Transfection......Page 70
3.1.2.4. Baculovirus Amplification......Page 71
3.2.1. Inverted Membrane Vesicles from the Selected Bacteria Strain......Page 72
3.2.3.1. Fluorescent-Substrates Transport Measurements......Page 73
3.2.3.2. ATPase Activity Measurements......Page 74
3.3.3. Purification of ABCG2 Overexpressed in High-Five Cells......Page 76
3.4.1. Quantification of Secondary Structure......Page 78
3.4.2. Ligand-Induced Quenching of Tryptophan Intrinsic Fluorescence......Page 80
3.5.1. Differential Solubilization of the Two ABCG2 Forms by Various Detergents......Page 81
3.5.2. Conversion of Lower into Upper Form of ABCG2 During SDS–PAGE in Presence of 9 M Urea......Page 83
3.5.3. Effects of Translation and Transcription Inhibitors on ABCG2 Expression in High-Five cells......Page 84
4. Notes......Page 85
Acknowledgments......Page 87
References......Page 88
Part II:
X-Ray Crystallography......Page 91
1. Introduction......Page 93
2. Materials......Page 94
3.1. Crystallization by the Vapor Diffusion Method......Page 95
3.2. Practical Approach to lipidic cubic phase (LCP) Crystallization......Page 97
3.3. Example of Bacteriorhodopsin (BR)......Page 98
3.4.1. Crystallization in the Sponge Phase......Page 100
3.4.2. Crystallization from Vesicles......Page 102
3.4.3. Crystallization from Bicelles......Page 103
3.4.4. On the Way to a General Method......Page 104
3.5.2. Crystallography......Page 105
4. Notes......Page 106
References......Page 114
1. Introduction......Page 119
2.1. General Strategy......Page 120
2.2. 3D Crystallization......Page 121
2.4. Description of the Structure......Page 122
2.5. Oligomerization, Organization in the Membrane, 2D Crystals......Page 124
3.1. What Is Suggested by the Structure?......Page 125
3.2. Molecular Dynamics Simulations on AAC......Page 126
3.3. Comparison and Differences with Other Transporters......Page 127
4. The Mitochondrial Carrier Family......Page 128
5. Conclusion......Page 129
References......Page 130
1. Introduction......Page 133
2. Use of Structural Analogues to Stabilize Intermediary Transport Steps......Page 136
3. Phosphorylation of Ca2+-ATPase by ATP......Page 138
4. Membrane Binding of Ca2+......Page 143
5. The Trans-location Step ((Ca2)E1P to E2P Transition)......Page 144
6. The Mechanism of E2-P Dephosphorylation......Page 147
7. The Dephosphorylated E2 and E1 States......Page 148
8. Concluding Comments......Page 150
References......Page 151
1. Introduction......Page 155
2.2.1. Protein Labeling......Page 158
2.2.2. Sample Setup......Page 159
3.1. Reconstitution of Protein in LCP......Page 160
3.1.2. Mixing Lipid with Protein......Page 161
3.2. LCP-FRAP Crystallization Pre-screening Assay......Page 162
3.2.1. Protein Labeling......Page 163
3.2.3. FRAP Data Collection......Page 164
3.2.4. Data Analysis......Page 166
3.3. Crystallization in Lipidic Mesophases......Page 167
3.3.1. Assembling Glass Sandwich Plates......Page 168
3.3.3. Crystal Detection......Page 169
3.3.4. Crystal Optimization......Page 170
3.3.5.2. Harvesting......Page 172
3.4.1. Alignment of Invisible Crystals with the Minibeam......Page 173
3.4.2. Crystallographic Data Collection......Page 174
5. Notes......Page 175
Acknowledgements......Page 180
References......Page 181
Part III:
Electron Microscopy......Page 184
1. Introduction......Page 185
2.2. Products......Page 190
3.1. Ultrastructural Cytology......Page 191
3.3. Negative Staining......Page 192
3.5. Gold Immunolabelling on Ultra-thin London Resin White Sections......Page 193
3.6. Gold Immunolabelling on Isolated Membranes......Page 194
3.8. Cryo-Electron Microscopy......Page 195
3.9. Image Processing......Page 196
4. Conclusions......Page 197
5. Notes......Page 198
References......Page 199
1. Introduction......Page 201
2.1. Analysis of Lipid......Page 202
2.4. Solid Carbon Films......Page 203
3. Methods......Page 204
3.1. Analysis of Lipid Species Copurifying with Membrane Proteins......Page 205
3.2. 2D Crystallization Using Biobeads......Page 206
3.3. 2D Crystallization by Detergent Removal......Page 207
3.4. Preparation of Solid Carbon Film for Electron Microscopy......Page 208
3.5. Preparation of Holey Carbon Films......Page 210
3.6. Preparation of Negatively Stained Samples......Page 211
3.7. Screening of 2D Crystallization Trials by Electron Microscopy......Page 212
4. Conclusion......Page 215
5. Notes......Page 216
References......Page 219
1. Introduction......Page 221
2. Materials......Page 223
3.2. Membrane Protein Reconstitution......Page 224
3.4. Tomogram Generation from Cryo-Tilt Series......Page 225
3.5. Single Particle Analysis......Page 228
4. Notes......Page 232
References......Page 233
1. Introduction......Page 235
2.2. Sample Preparation for Electron Microscopy......Page 236
3.1. Sample Preparation......Page 237
3.2. Images Acquisition......Page 238
3.3.1. Image Alignment......Page 240
3.3.2. Tomograms Reconstructions......Page 242
3.4. Conclusions......Page 245
4. Notes......Page 247
References......Page 249
1. Introduction......Page 251
2. Materials......Page 253
3.1. Flexible Fitting of Atomic Structures into an EM Map of an Isolated Protein......Page 254
3.2. Analysis of the Fitting Procedure as a Conformational Transition......Page 258
3.3. Analysis of the Fitted Structures......Page 261
3.4. Localization of Intramolecular Decavanadate......Page 264
3.5. Localization of Intermolecular Decavanadate After Tubular Reconstruction......Page 266
4. Conclusions......Page 267
5. Notes......Page 269
References......Page 270
Part IV:
Nuclear Magnetic Resonance......Page 274
1. Introduction......Page 275
2.1. Over-expression......Page 277
2.2. Tag......Page 278
3.1.1.1. Choice of Detergent/Phopholipids......Page 279
3.1.1.2. Labelling......Page 280
3.1.2.2. Backbone Chemical Shifts Assignments......Page 281
3.1.2.3. Long Range Restraints......Page 283
3.1.2.4. Residual Dipolar Couplings (RDCs)......Page 284
3.1.2.6. Toward MP Dynamics and Mechanisms......Page 285
3.2. Solid-State NMR of Membrane Proteins......Page 286
3.2.1.1. Aligned Samples......Page 288
3.2.1.2. Resonance Assignments and Structure Determination......Page 289
3.2.2.1. Sample Preparation......Page 290
3.2.2.2. Resonance Assignments......Page 291
4. Conclusion and Perspectives......Page 292
References......Page 293
1. Introduction......Page 297
3.1. Secondary Structures of Fragments of Membrane Proteins Corresponding to Single Loops or to Single Transmembrane Helices......Page 300
3.1.1. Transmembrane Helical Fragments......Page 301
3.1.2. Fragments Corresponding to Loops of Membrane Proteins......Page 302
3.1.3. General Features of Membrane Protein Structure Accessible from Studies of Membrane Protein Fragments......Page 305
3.2. Two-Helix Fragments......Page 306
3.3. Full Scan of Secondary Structure Using Membrane Protein Fragments......Page 307
3.4. Three-Dimensional Structures from Protein Fragments......Page 309
3.5. NMR Studies on Intact Membrane Proteins......Page 311
References......Page 312
1. Introduction......Page 317
2.1.3. Purification of PLN......Page 319
2.4.1. Solution-State Samples for Titration......Page 320
3.1.2. PLN Expression......Page 321
3.1.3. PLN Purification......Page 322
3.3.2. ATPase Activity Measurements......Page 323
3.3.3. 31P NMR Activity Assays......Page 324
3.4.2. Kd Measurements by NMR......Page 325
3.4.3. Preparation of Oriented Samples and Solid-State NMR Spectroscopy......Page 326
3.4.4. Structure Determination of Monomeric PLN Using a Hybrid Solution and Solid-State NMR Method......Page 327
3.5.1. Allosteric Model of SERCA Regulation by PLN......Page 328
3.5.2. Rational Design of PLN Mutants as Possible Candidates for Gene Therapy......Page 329
4. Notes......Page 330
References......Page 331
1. Introduction......Page 335
2.1. Cells Culture and Isotope Labelling......Page 336
2.3. Detergent Screening and NMR Sample Preparation......Page 337
3.1.1. Uniform (2H,13C,15N) Labelling......Page 338
3.1.3. Fractional 13C (10%) labelling......Page 339
3.2. In Vitro Refolding from E. coli Inclusion Bodies......Page 340
3.3. Detergent Screening and NMR Sample Preparation......Page 341
3.4. Solution-State NMR Experiments for Resonance Assignments and Structure Determination......Page 342
3.4.2. Measurement of Backbone HN–HN NOEs Using a 4D 15N,15N Separated NOESY-TROSY Experiment......Page 343
3.4.3. Selective Methyl Protonation Approach to Structure Determination......Page 346
3.5. Structure Calculation......Page 349
4. Notes......Page 351
References......Page 352
1. Introduction......Page 355
2.1. Samples, Preparation Tools and Measuring Cells......Page 356
2.5. NMR Instrumentation for HR-MAS 1H-NMR......Page 357
3. Methods......Page 358
3.1. Determination of Lipid Phase Nature (Lamellar (Liposomes), Oriented-Bicelle, Isotropic) Using Wide Line 31P-NMR......Page 361
3.3. Determination of Lipid Dynamics in Non-oriented Multilamellar Bilayers (Gel, Fluid, Liquid-Ordered) Using Wide Line 2H......Page 364
3.4. Determination of Lipid Molecular Structure in a Membrane Environment Using 1H HR-MAS (Fig. 3)......Page 366
3.5. Determination of Lipid Internal Dynamics in Oriented Bilayers Using 2H-NMR Spin-Lattice Relaxation Time Measurements......Page 369
4. Notes......Page 371
References......Page 372
Part V:
Molecular Modelling......Page 375
1. Introduction......Page 377
3.1.1. With Available Known Homologues......Page 379
3.1.2. Without Known Homologues......Page 382
3.2.1.2. Helices Conformation and Relative Positioning......Page 384
3.2.2.1. Molecular Dynamics Simulations and Related Approaches......Page 386
3.2.2.2. Normal Mode Analysis......Page 388
3.3. Conclusion......Page 389
4. Notes......Page 390
References......Page 396
1. Introduction......Page 401
3. Methods......Page 402
3.1. Sequence Analyses: Multiple Sequence Alignment and Bibliographic Researches......Page 405
3.2. Of Helices and Transmembrane Segments......Page 407
3.3. Comparative Modeling and Other Approaches......Page 408
3.5. Helix–Helix Interactions......Page 411
3.7. Of Loops and Mutants......Page 412
4. Notes......Page 413
Acknowledgments......Page 414
References......Page 415
1. Introduction......Page 417
2.1. Computers......Page 418
2.3. Software......Page 419
2.6. Simulation Parameters......Page 420
3.1. General Considerations on the Comparison of MD to Experiments......Page 422
3.2. Comparison of MD to Biochemical Data......Page 423
3.3. Comparison of MD to Biophysical Data......Page 425
3.4. One Guided Example: Comparison of MD Simulations to 2H NMR Data on a Transmembrane Peptide......Page 427
4. Notes......Page 431
References......Page 433
1.1. Membranes and Membrane Proteins......Page 437
2.1. Molecular Modeling......Page 438
2.2. Quantum Mechanics......Page 439
2.4. Limitations of Computer Simulations......Page 441
2.5. Applications......Page 442
2.5.2.1. G Protein-Coupled Receptors......Page 443
25.2.2. Transport Across the Membrane: Transporters and Channels......Page 444
2.5.2.4. Channels......Page 445
2.5.2.5. Ion Channels......Page 446
2.5.3.2. Protein Aggregation and Protein-Membrane Self-Assembly......Page 447
2.5.4. Beyond the Microsecond......Page 449
Acknowledgments......Page 450
References......Page 451
1. Introduction......Page 455
3.1. Mapping of CCK1R Binding Site: Synergy Between Laboratory and In Silico Experiments......Page 457
3.2. Local GPCR Drug Design......Page 462
3.3. Global GPCRs Drug Design......Page 464
References......Page 466
Index......Page 469