title: Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences.
abstract: Amphipols are short amphipathic polymers designed to stabilize membrane proteins in aqueous solutions in the absence of detergent. Bacteriorhodopsin (BR), a light-driven proton pump, has been denatured, either by direct solubilization of the purple membrane in sodium dodecylsulfate (SDS) solution or by a procedure that involves delipidation with organic solvent followed by transfer to SDS, and renatured in amphipol A8-35. The effect of different renaturation procedures and of the presence or absence of lipids and the cofactor retinal have been investigated. The resulting samples have been characterized by absorbance spectroscopy, size-exclusion chromatography, thermostability measurements, and determination of photocycle kinetics. Transfer to A8-35 can be achieved by SDS precipitation, dilution, or dialysis, the first route resulting in the highest yield of refolding. Functional BR can be refolded whether in the presence or absence of lipids, higher yields being achieved in their presence. Retinal is not required for the protein to refold, but it stabilizes the refolded form and, thereby, improves folding yields. Lipids are not required for BR to perform its complete photocycle, but their presence speeds up the return to the ground state. Taken together, these data indicate that a membrane or membrane-mimetic environment is not required for correct decoding of the chemical information contained in the sequence of BR; functional folding is possible even in the highly foreign environment of lipid-free amphipols. BR interactions with lipids, however, contribute to an effective photocycle.
authors.affiliation: UMR 7099, CNRS/Université Paris-7, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.
authors.forename: Tassadite
authors.initials: T
authors.lastname: Dahmane
authors.forename: Fabrice
authors.initials: F
authors.lastname: Rappaport
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Popot
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D008563:Membrane Lipids / Q000494:pharmacology*; D011108:Polymers / Q000494:pharmacology*; D011437:Propylamines / Q000494:pharmacology*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects*; D058849:Protein Refolding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D012172:Retinaldehyde / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D008563:Membrane Lipids; D011108:Polymers; D011437:Propylamines; D012997:Solvents; C499017:amphipol A8-35; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D012172:Retinaldehyde