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 MP? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
xx
Contents
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 MP? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
xx
Contents