Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10156934 | Methods | 2018 | 42 Pages |
Abstract
Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called 'amphipols' (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both α-helical and β-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.
Keywords
CFEIMACPDSIBSGPCRCryo-EMGdnHClCACnative conformationBacterio-opsinSECThree-dimensionalOTGguanidine chlorideCMCSDSCa2+-ATPaseAmphipolSDS-polyacrylamide gel electrophoresisSDS-PAGERefoldingCell-free expressionOverexpressionStabilityMolecular dynamicscircular dichroismsodium dodecylsulfateSurfactantscritical aggregation concentrationcritical micelle concentrationtransmembraneInclusion bodiesMolecular weightMembrane proteinSize exclusion chromatographyImmobilized-metal affinity chromatographyG protein-coupled receptor
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Biochemistry
Authors
Christel Le Bon, Anaïs Marconnet, Sandrine Masscheleyn, Jean-Luc Popot, Manuela Zoonens,