Membrane proteins in aqueous solutions : from detergents to amphipols

목차 (Table of Contents)

• CONTENTS
• 1 Membrane Proteins and Their Natural Environment = 1
• 1.1 Introduction = 1
• 1.2 Lipid Bilayers = 4
• 1.3 Membrane Protein Functions = 8
• 1.4 Membrane Protein Structure = 13
• 1.4.1 Modes of Association with the Membrane = 13
• 1.4.2 Structure of Transmembrane Protein Regions = 15
• 1.5 Membrane Protein / Lipid Interactions = 22
• 1.5.1 The Fluid Mosaic Model = 22
• 1.5.2 Bound Lipids = 25
• 1.6 Dynamics of Transmembrane Regions and the Function of Membrane Proteins = 32
• 1.6.1 Bacteriorhodopsin = 32
• 1.6.2 The Nicotinic Acetylcholine Receptor = 36
• 1.6.3 The Sarcoplasmic Reticulum Calcium Pump = 44
• 1.7 Synthesis of Membrane Proteins = 48
• 1.7.1 Natural Biosynthesis = 48
• 1.7.2 Overexpression = 50
• References = 51
• 2 Extracting Membrane Proteins from Their Native Environment = 59
• 2.1 Introduction = 59
• 2.2 Detergents = 60
• 2.2.1 Chemical Structure = 60
• 2.2.2 Physical-Chemical Properties = 62
• 2.3 Solubilizing Membrane Proteins with Detergents = 65
• 2.3.1 Solubilizing Biological Membranes with Detergents = 65
• 2.3.2 Membrane Protein / Detergent Complexes = 68
• 2.4 Why Are Membrane Proteins Unstable in Detergent Solutions? = 70
• 2.4.1 Instability of Detergent-Solubilized Membrane Proteins Is a General Phenomenon = 70
• 2.4.2 A Field Case : Inactivation of the Cytochrome b6f Complex by Detergents = 72
• 2.4.3 The Dissociating Character of Detergents as a Major Cause of Membrane Protein Inactivation = 76
• 2.4.4 Detergent-Induced Conformational Alterations = 80
• 2.5 Solutions to the Instability Problem = 81
• 2.5.1 Making Detergents Less Aggressive = 81
• 2.5.2 Making Membrane Proteins More Resistant = 84
• 2.5.3 What About Keeping Membrane Proteins Water-Soluble Without Using Detergents? = 87
• References = 88
• 3 Alternatives to Detergents for Handling Membrane Proteins in Aqueous Solutions = 97
• 3.1 Introduction = 97
• 3.2 Bicelles = 99
• 3.2.1 The Formation and Phase Diagram of Bicelles and Their Use in Membrane Protein NMR Spectroscopy = 99
• 3.2.2 Bicelles and Membrane Protein Crystallography = 103
• 3.2.3 Other Applications of Bicelles in Membrane Biology = 104
• 3.3 Nanodiscs = 105
• 3.3.1 High-Density Lipoproteins = 105
• 3.3.2 The Formation and Structure of Nanodiscs = 107
• 3.3.3 The Empty Nanodisc = 109
• 3.3.4 Membrane Protein／Nanodisc Complexes = 111
• 3.3.5 Nanodisc-Based Investigations of Membrane Proteins = 115
• 3.4 Amphipathic Peptides = 118
• 3.4.1 Peptitergents = 120
• 3.4.2 Lipopeptide Detergents = 120
• 3.4.3 Designer Peptide Surfactants (Peptergents) = 121
• 3.4.4 Stabilizing Membrane Proteins by Complexation with Saposin A ("Picodiscs," "Sap A discs," "Salipro® Nanoparticles") = 123
• 3.4.5 Peptide-Based Nanodiscs = 127
• 3.5 Fluorinated Surfactants = 128
• 3.5.1 Background = 128
• 3.5.2 Novel Structures of Fluorinated Surfactants = 130
• 3.5.3 Applications of Fluorinated Surfactants = 131
• 3.6 Nonconventional Surfactants Used for Handling Membrane Proteins in Aqueous Solutions : An Overview = 133
• References = 135
• 4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols = 151
• 4.1 Introduction = 151
• 4.2 Amphipol Chemical Structure and Synthesis = 152
• 4.2.1 Polyacrylate-Based Amphipols with Carboxylates as Their Hydrophilic Moieties = 153
• 4.2.2 Other Ionic Amphipols = 162
• 4.2.2.1 Sulfonated Amphipols = 162
• 4.2.2.2 Phosphorylcholine-Based Amphipols = 162
• 4.2.2.3 PMAL Series = 163
• 4.2.2.4 Styrene-Maleic Acid Copolymer = 163
• 4.2.2.5 Hydrophobically Grafted Poly-γ-Glutamic Acid (APG) = 164
• 4.2.3 Non-ionic Amphipols = 165
• 4.2.3.1 THAM-Based Non-ionic Amphipols = 165
• 4.2.3.2 Glucose-Based Non-ionic Amphipols (NAPols) = 167
• 4.2.3.3 NVoy = NV 10 = 169
• 4.3 Self-Association Behavior of Amphipols in Aqueous Solutions = 169
• 4.3.1 Formation, Structure, and Dynamics of A8-35 Particles = 170
• 4.3.1.1 Critical Association Concentration = 170
• 4.3.1.2 The Size, Shape, and Organization of A8-35 Particles = 176
• 4.3.1.2.1 Mass and Dispersity of Individual A8-35 Molecules = 176
• 4.3.1.2.2 Homogeneity, Mass, and Size of A8-35 Particles = 178
• 4.3.1.2.3 Shape and Internal Organization of A8-35 Particles = 183
• 4.3.1.2.4 Dynamics of A8-35 Particles = 187
• 4.3.1.2.5 Data Pertaining to the Phase Diagram of A8-35 = 191
• 4.3.2 Solution Properties of Other Amphipols = 192
• 4.4 Labeled and Functionalized Amphipols = 198
• 4.4.1 Synthesis of Labeled or Tagged Derivatives of A8-35 and A8-75 = 198
• 4.4.2 Solution Behavior of Labeled or Tagged Derivatives of A8-35 and A8-75 = 203
• 4.5 Protocol 4.1 : Synthesis of A8-35 = 205
• 4.5.1 Preparation and Characterization of the PAA Precursor = 205
• 4.5.2 Hydrophobic Modification of the PAA Precursor = 207
• 4.5.3 Purification of A8-35 = 208
• 4.5.4 Determination of the Chemical Composition of the Final Product = 208
• 4.6 Annexes = 210
• 4.6.1 Annex 4.1. Determining and Expressing the Average Mass and Dispersity of Polymers = 210
• 4.6.1.1 The Origin of Dispersity = 210
• 4.6.1.2 How Can Size Dispersity Be Limited? = 211
• 4.6.1.3 Expressing the Average Mass and Dispersity of Polymers = 213
• 4.6.1.4 Determining the Average Mass and Dispersity of Polymers = 214
• 4.6.1.5 Calibration of Size Exclusion Chromatography Columns = 214
• 4.6.2 Annex 4.2. Kinetics of Conventional Radical Polymerization and Origin of Size Dispersity = 216
• 4.6.3 Annex 4.3. Expression of the Instantaneous and Cumulative Average Degree of Polymerization in Conventional Radical Polymerization = 217
• 4.6.4 Annex 4.4. How to Control the Size of Polymers Obtained by Radical Polymerization? = 218
• 4.6.4.1 Telomerization (Chain Transfer) = 218
• 4.6.4.2 Controlled / Living Radical Polymerization (CRP) = 219
• 4.6.5 Annex 4.5. Polymer Topology and How to Control It = 222
• 4.6.5.1 Polymer Topology = 222
• 4.6.5.2 Polymer Composition (Microstructure) = 223
• 4.6.6 Annex 4.6. Functionalizing Polymers = 225
• 4.6.6.1 General Considerations = 225
• 4.6.6.2 Possible Applications of Controlled Radical Polymerization to the Chemistry of Amphipols = 227
• References = 227
• 5 Formation and Properties of Membrane Protein／Amphipol Complexes = 237
• 5.1 Introduction = 237
• 5.2 Forming Membrane Protein／Amphipol Complexes = 238
• 5.2.1 Transferring Native Membrane Proteins from Detergent Solution to Amphipols = 252
• 5.2.2 Direct Extraction of Proteins from Membranes = 259
• 5.2.2.1 Styrene-Maleic Acid Copolymers = 260
• 5.2.2.2 Can Membrane Proteins Be Directly Solubilized Using Polyacrylate-Based Amphipols? = 261
• 5.2.3 Folding Membrane Proteins in Amphipols = 265
• 5.3 Composition, Organization, Dynamics, and Solution Properties of Membrane Protein／Amphipol Complexes = 266
• 5.3.1 Particle Composition = 273
• 5.3.1.1 Amphipol vs. Detergent Binding = 274
• 5.3.1.2 Lipid Binding = 275
• 5.3.2 Particle Size and Organization = 279
• 5.3.3 Protein／Polymer Interactions = 283
• 5.4 Functionality of Amphipol-Trapped Membrane Proteins = 286
• 5.5 Biochemical Stability of Amphipol-Trapped Membrane Proteins = 290
• 5.6 Membrane Protein Dynamics and the Effects of Amphipols on Stability and Function = 296
• 5.6.1 Functional Observations = 297
• 5.6.2 Molecular Dynamics Simulations = 303
• 5.7 Transferring Membrane Proteins from Amphipols to Other Environments = 306
• 5.8 Conclusion = 310
• 5.9 Protocols = 311
• 5.9.1 Protocol 5.1. Transferring MPs from Detergents to APols = 311
• 5.9.1.1 Preparation of a Stock Solution of APols = 311
• 5.9.1.2 Determination of the Protein Concentration = 311
• 5.9.1.3 Determination of the Optimal MP/APol Mass Ratio = 312
• 5.9.1.4 Detergent Removal = 312
• 5.9.1.5 Identification of the Optimal MP/APol Ratio = 313
• 5.9.2 Protocol 5.2. Determining the Amount of MP-Bound APol = 314
• 5.9.2.1 Why Is It Preferable to Express the Amount of APols Bound per MP in Mass Rather Than as a Number of Molecules? = 315
• 5.9.2.2 How to Estimate A Priori the Likely Amount of APols Bound per MP Based on Structural Data? = 315
• 5.9.2.3 How to Experimentally Measure the Quantity of APols Bound per MI39 = 316
• 5.9.3 Protocol 5.3. Transferring a MP from A8-35 to Nanodiscs = 319
• 5.9.3.1 Exchange of A8-35 for DDM = 319
• 5.9.3.2 Reconstitution into Nanodiscs = 320
• References = 322
• 6 Amphipol-Assisted Folding of Membrane Proteins = 333
• 6.1 Introduction = 333
• 6.2 Context : Existing Approaches to Folding Membrane Proteins In Vitro = 334
• 6.3 Amphipol-Assisted Folding of Membrane Proteins = 338
• 6.3.1 Which Membrane Proteins Have Been Folded in Amphipols and How = 338
• 6.3.1.1 Amphipol-Assisted Folding of α-Helical Membrane Proteins = 339
• 6.3.1.1.1 Folding of BR in A8-35 = 339
• 6.3.1.1.2 A8-35-Assisted Folding of GPCRs = 343
• 6.3.1.1.3 Folding of α-Helical Membrane Proteins in Non-ionic Amphipols = 345
• 6.3.1.2 Amphipol-Assisted Folding of β-Barrel Membrane Proteins = 346
• 6.3.1.2.1 Folding of OmpA and FomA in A8-35 = 347
• 6.3.1.2.2 Folding of tOmpA, OmpT, and PagP = 348
• 6.3.1.2.3 Kinetics and Thermodynamics of Folding OmpA in A8-35 = 349
• 6.3.2 Why Are Amphipols a Good Medium for Membrane Protein Folding? = 350
• 6.3.3 Challenges and Prospects = 353
• 6.4 Protocol 6.1. Amphipol-Assisted Folding of Membrane Proteins = 354
• 6.4.1 Solubilization and Purification of MPs in Denaturing Conditions = 354
• 6.4.2 Renaturation of α-Helical MPs in APols = 355
• 6.4.3 Renaturation of β-Barrel MPs in APols = 356
• 6.4.4 Completing the Renaturation = 356
• References = 356
• 7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins = 361
• 7.1 Introduction = 361
• 7.2 Context : Cell-Free Expression of Membrane Proteins = 363
• 7.3 Cell-Free Expression of Membrane Proteins Using Amphipols and Other Amphipathic Polymers = 365
• 7.4 Conclusions and Perspectives = 373
• 7.5 Protocol 7.1 : Cell-Free Expression of Membrane Proteins in the Presence of Non-ionic Amphipols = 375
• 7.5.1 Introduction = 375
• 7.5.2 Protocol = 375
• References = 376
• 8 Optical Spectroscopy of Membrane Protein / Amphipol Complexes = 381
• 8.1 Introduction = 381
• 8.2 Optical Spectroscopy Studies of Amphipols and Membrane Protein / Amphipol Complexes = 382
• 8.2.1 Circular Dichroism and Synchrotron Radiation Circular Dichroism = 391
• 8.2.2 Infrared and Raman Spectroscopy = 394
• 8.2.3 Fluorescence Spectroscopy = 397
• 8.3 Conclusions and Prospects = 400
• References = 401
• 9 Solution Studies of Membrane Protein / Amphipol Complexes = 405
• 9.1 Introduction = 406
• 9.2 Overview of the Literature = 409
• 9.3 A Walk Through Selected Solution Studies = 410
• 9.3.1 Sucrose Gradient Rate Zonal Ultracentrifugation = 411
• 9.3.2 Size Exclusion Chromatography = 417
• 9.3.3 Blue-Native Polyacrylamide Gel Electrophoresis = 419
• 9.3.4 Chemical Cross-Linking = 421
• 9.3.5 Electron Resonance Spectroscopy = 421
• 9.3.6 Determination of Rotational Correlation Times by Solution NMR = 422
• 9.3.7 Analytical Ultracentrifugation = 423
• 9.3.8 Small-Angle X-ray and Neutron Solution Scattering = 426
• 9.3.8.1 Introduction = 426
• 9.3.8.2 Small-Angle Neutron Scattering = 432
• 9.3.8.3 Small-Angle X-ray Scattering = 437
• 9.3.9 Dynamic Light Scattering = 438
• 9.4 Conclusions and Prospects = 439
• 9.5 Protocols = 440
• 9.5.1 Protocol 9.1 : Analytical Ultracentrifugation of Membrane Protein / Amphipol Complexes = 440
• 9.5.1.1 Introduction = 440
• 9.5.1.2 Instrumentation = 441
• 9.5.1.3 Designing the Samples and Experiments = 441
• 9.5.1.4 Compiling the Parameters That Will Be Required for the Analysis = 442
• 9.5.1.5 Running SV Experiments = 442
• 9.5.1.6 c(s) Analysis = 442
• 9.5.2 Protocol 9.2 : Size Exclusion Chromatography Analysis of Membrane Protein / Amphipol Complexes = 444
• 9.5.2.1 Introduction = 444
• 9.5.2.2 Biochemistry = 444
• 9.5.2.3 Size Exclusion Chromatography System = 445
• 9.5.2.4 Buffers = 445
• 9.5.2.5 Protocol = 446
• 9.5.2.6 Commented Examples = 446
• References = 448
• 10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins = 453
• 10.1 Introduction = 453
• 10.2 Context : Solution NMR Studies of Membrane Proteins = 455
• 10.2.1 Producing the Protein = 455
• 10.2.2 Membrane Protein / Surfactant Complexes Suitable for Solution NMR = 456
• 10.3 Solution NMR Studies of Amphipol-Trapped Membrane Proteins = 461
• 10.3.1 Validating and Developing the Methodology = 463
• 10.3.1.1 Validating the Use of Amphipols for sNMR and Resolution of the Spectra as Compared with Those Obtained in Detergent Solutions = 465
• 10.3.1.2 Mapping Membrane Protein / Amphipol Interactions by NMR = 469
• 10.3.2 Developing pH-Insensitive and Fully Deuterated Amphipols = 473
• 10.3.2.1 pH-Insensitive Amphipols and Their Use in sNMR = 473
• 10.3.2.2 Deuterated Amphipols = 479
• 10.3.3 Biologically Oriented Studies = 482
• 10.3.3.1 Dynamics of the Transmembrane β-Barrel of OmpX = 483
• 10.3.3.2 Conformation of LTB4 Bound to the BLT2 Leukotriene Receptor = 483
• 10.3.3.3 Preliminary Studies of Chlamydia trachomatis MOMP and Two Melanocortin Receptors = 488
• 10.4 Challenges and Prospects = 488
• References = 490
• 11 Amphipols and Membrane Protein Crystallization = 497
• 11.1 Introduction = 497
• 11.2 Context : Classical and Less Classical Approaches to Crystallizing Membrane Proteins = 498
• 11.2.1 Factors Contributing to the Steady Growth of the Number of MP Structures Solved = 498
• 11.2.2 The Formation of Membrane Protein Crystals = 500
• 11.2.2.1 Crystals Formed in Detergent Solutions = 503
• 11.2.2.2 Forming Crystals in Lipid Three-Dimensional Phases = 506
• 11.3 Using Amphipols for Crystallizing Membrane Proteins = 508
• 11.3.1 Crystallization of Cytochrome bc1 / A8-35 Complexes in Aqueous Solutions : Failures and Partial Successes and the Possible Reasons Underlying Them = 509
• 11.3.2 Using Amphipols as a Vehicle to Crystallize Membrane Proteins in Meso = 516
• 11.3.3 Challenges and Prospects = 522
• 11.4 Protocol: Crystallizing bc1 / A8-35 / Detergent Complexes from Aqueous Solutions = 523
• 11.4.1 Pre-crystallization = 523
• 11.4.2 Crystallization of Ternary Protein / Amphipol/Detergent Complexes = 524
• References = 525
• 12 The Use of Amphipols for Electron Microscopy = 533
• 12.1 Introduction = 533
• 12.2 Context : Electron Microscopy Studies of Membrane Proteins = 534
• 12.2.1 The Various Approaches to Studying Membrane Proteins by Electron Microscopy = 534
• 12.2.2 Single-Particle Electron Microscopy Studies of Biological Molecules : The Basics = 535
• 12.2.3 Single-Particle Cryo-EM Studies of Biological Molecules : The Resolution Revolution = 538
• 12.2.4 Single-Particle Electron Microscopy Studies of Membrane Proteins = 540
• 12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies = 541
• 12.3.1 The Early Years : 1998-2008 = 549
• 12.3.2 Recent Studies of Negatively Stained MP / APol Complexes (2011-Present) = 554
• 12.3.2.1 Conclusions from Negative-Stain Studies = 561
• 12.3.3 Cryo-EM Studies of MP / APol Complexes (2011-Present) = 562
• 12.3.4 Conclusions and Perspectives = 573
• 12.3.4.1 Solution Properties of the Samples = 573
• 12.3.4.2 Imaging = 574
• 12.3.4.3 Perspectives, Suggestions, and Speculations = 577
• 12.4 Protocol 12.1 : Preparation of Amphipol-Trapped Membrane Proteins for Cryo-EM Studies = 579
• 12.4.1 Introduction = 579
• 12.4.2 Vectors and Cell Lines = 580
• 12.4.3 Buffers = 580
• 12.4.4 Protocol = 581
• References = 583
• 13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications = 591
• 13.1 Introduction = 591
• 13.2 Immobilization of Membrane Proteins Using Biotinylated Amphipols and Its Applications = 595
• 13.2.1 Validation of the Approach Using Biotinylated A8-35 = 595
• 13.2.2 Multiplexing = 600
• 13.2.3 Using Ionic vs. Non-ionic Biotinylated Amphipols for Phage Display Selection of Membrane Protein Binders = 604
• 13.3 Alternative Tags = 607
• 13.3.1 Polyhistidine-Tagged A8-35 = 608
• 13.3.2 Imidazole-Tagged Amphipols = 611
• 13.3.3 Oligonucleotide-Tagged Amphipol = 613
• 13.3.4 Sulfide- and Sulfhydryl-Tagged Amphipols = 615
• 13.4 Other Applications of Tagged Amphipols = 616
• 13.5 Protocol 13.1. Amphipol-Mediated Immobilization of Membrane Proteins for Surface Plasmon Resonance Experiments = 617
• 13.5.1 Experimental Setup = 617
• 13.5.2 Preparation of Samples = 618
• 13.5.3 Measurements = 618
• References = 619
• 14 The Use of Amphipols in Mass Spectrometry = 625
• 14.1 Introduction = 625
• 14.2 MALDI-TOF Studies = 633
• 14.3 Electrospray Ionization-Mass Spectrometry Studies = 636
• 14.3.1 Membrane Protein / Amphipol Complexes Are Amenable to Electrospray Mass Spectrometry = 636
• 14.3.2 Amphipols, ESI-MS, and Oligomeric Membrane Proteins = 640
• 14.3.3 Interactions of Detergents vs. Amphipols with Membrane Proteins as Probed by Mass Spectrometry = 641
• 14.3.4 Can Amphipols Better Suited to ESI-MS Be Developed? = 643
• 14.4 Proteomics = 644
• 14.5 Protocol 14.1 : Characterization of Amphipol-Trapped Membrane Proteins by Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry = 648
• 14.5.1 Introduction = 648
• 14.5.2 Materials = 648
• 14.5.3 Protocol = 649
• 14.5.4 Outlook = 654
• References = 655
• 15 Biomedical Applications = 659
• 15.1 Introduction = 659
• 15.2 Using Amphipols to Formulate a Subunit Vaccine Against Chlamydia trachomatis = 661
• 15.2.1 Background = 661
• 15.2.2 Developing an Amphipol-Based Vaccine = 664
• 15.2.3 How Could Amphipols Help in Formulating More Efficient Vaccines? = 669
• 15.3 Uptake of A8-35 and a Passenger Peptide by Cells in Culture = 672
• 15.4 Biodistribution and Elimination of Amphipols Following Injection in Mice = 674
• References = 678
• Final Comments = 683
• Index = 687