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Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences

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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.

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Acknowledgments

Special thanks are due to Frank Wien (Synchrotron Soleil, Saclay, France) for analyzing samples on the circular dichroism synchrotron beamline Bessy (Helmholtz-Zentrum, Berlin), to Manuela Zoonens (UMR 7099) for her contribution to collecting UV–visible spectra, to Fabrice Giusti (UMR 7099) for the synthesis of amphipol A8-35, and to J. Barra for her kind help with preparation of the figures. This work was supported by the CNRS, by University Paris-7, and by grants from the E.U. (Specific Targeted Research Project LSHGCT-2005-513770 IMPS: Innovative tools for membrane protein structural proteomics) and from the French Ministry of Research (ANR-06-BLAN-0087 Refolding GPCRs). T.D. was the recipient of a fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche.

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  1. Tassadite Dahmane

    Present address: Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, 202 BB, New York, NY, 10032, USA

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  1. UMR 7099, CNRS/Université Paris-7, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France

    Tassadite Dahmane & Jean-Luc Popot

  2. UMR 7141, CNRS/Université Paris-6, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France

    Fabrice Rappaport

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  1. Tassadite Dahmane
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Correspondence to Jean-Luc Popot.

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Special Issue: Structure, function, folding, and assembly of membrane proteins—insight from biophysics.

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Dahmane, T., Rappaport, F. & Popot, JL. Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences. Eur Biophys J 42, 85–101 (2013). https://doi.org/10.1007/s00249-012-0839-z

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