Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Author:

Higgins Anna J.,Flynn Alex J.,Marconnet Anaïs,Musgrove Laura J.,Postis Vincent L. G.,Lippiat Jonathan D.ORCID,Chung Chun-wa,Ceska TomORCID,Zoonens ManuelaORCID,Sobott FrankORCID,Muench Stephen P.ORCID

Abstract

AbstractMembrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.

Funder

RCUK | Biotechnology and Biological Sciences Research Council

Publisher

Springer Science and Business Media LLC

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

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