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

TY - JOUR

T1 - Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

AU - Higgins, Anna J.

AU - Flynn, Alex J.

AU - Marconnet, Anaïs

AU - Musgrove, Laura J.

AU - Postis, Vincent L. G.

AU - Lippiat, Jonathan D.

AU - Chung, Chun-wa

AU - Ceska, Tom

AU - Zoonens, Manuela

AU - Sobott, Frank

AU - Muench, Stephen P.

N1 - Funding Information: We would like to acknowledge the Muench and Sobott labs for fruitful discussions, particularly Anton Calabrese and David Klebl. The authors thank Arndt Rohwedder for assistance with TLC, and the Astbury Biostructure Laboratory for their assistance with EM data collection. The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust (108466/Z/15/Z). This work and A.J.H. and L.J.M. were funded through a BBSRC grant (BB/R018561/1/). The amphipol development in IBPC Paris was supported by the Centre National de la Recherche Scientifique (CNRS), Université de Paris (Université Paris 7), and the ‘Initiative d’Excellence’ programme from the French State (Grant ‘DYNAMO’, ANR-11-LABX-0011-01). Publisher Copyright: © 2021, The Author(s).

PY - 2021/11/25

Y1 - 2021/11/25

N2 - Membrane 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.

AB - Membrane 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.

KW - Cryoelectron microscopy

KW - Membranes

UR - https://www.scopus.com/pages/publications/85119852881

U2 - 10.1038/s42003-021-02834-3

DO - 10.1038/s42003-021-02834-3

M3 - Article

C2 - 34824357

AN - SCOPUS:85119852881

SN - 2399-3642

VL - 4

JO - Communications Biology

JF - Communications Biology

M1 - 1337

ER -