Folding and stabilizing membrane proteins in amphipol A8-35

• Published in: Methods (San Diego, Calif.) Calif.), 2018-09, Vol.147, p.95-105
• Main Authors: Le Bon, Christel, Marconnet, Anaïs, Masscheleyn, Sandrine, Popot, Jean-Luc, Zoonens, Manuela
• Format: Article
• Language: English
• Subjects: Biochemistry, Molecular Biology; Inclusion bodies; Life Sciences; Membrane Proteins - chemistry; Native conformation; Overexpression; Polymers - chemistry; Propylamines - chemistry; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Protein Stability; Refolding; Stability; Surfactants
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2018.04.012

•Amphipathic polymers (amphipols) are relevant tools for membrane protein studies.•Folding membrane proteins overexpressed in inclusion bodies using amphipols.•Replacing detergents by amphipols increases the stability of membrane proteins. 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.