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

TY - GEN

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

AU - Higgins, Anna

AU - Flynn, Alex

AU - Marconnet, Anaïs

AU - Musgrove, Laura

AU - Postis, Vincent

AU - Lippiat, Jonathan

AU - Chung, Chun-Wa

AU - Ceska, Tom

AU - Zoonens, Manuela

AU - Sobott, Frank

AU - Muench, Stephen

N1 - 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 AJH was 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” program from the French State (Grant “DYNAMO”, ANR-11-LABX-0011-01)

PY - 2021/1/15

Y1 - 2021/1/15

N2 - Membrane proteins are essential for cellular growth and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant 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 significant advantage. Here we use a novel 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 detergent-free 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 and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant 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 significant advantage. Here we use a novel 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 detergent-free approach for the study of membrane proteins allowing native extraction and high-resolution structure determination by cryoEM.

KW - CryoEM

KW - membrane protein

KW - amphipol

KW - detergent

UR - http://europepmc.org/abstract/PPR/PPR266018

U2 - 10.21203/rs.3.rs-131488/v1

DO - 10.21203/rs.3.rs-131488/v1

M3 - Other contribution

PB - Research Square

ER -