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

Anna J Higgins,Chun-Wa Chung,Vincent L G Postis,Tom Ceska,Jonathan D Lippiat,Frank Sobott,Anaïs Marconnet,Alex J Flynn,Laura J Musgrove,Stephen P Muench,Manuela Zoonens

doi:10.1038/s42003-021-02834-3

Abstract

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.

Highlights

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets
It was determined that a 0.1% (w/v) concentration of CyclAPols was sufficient for downstream experiments, with yields only slightly lower relative to that obtained for the styrene and maleic acid (SMA) polymer, despite the lower polymer concentration for CyclAPols (0.1 vs 1%)
Classical APols such as A8-35 are effective in cryo-electron microscopy (cryoEM), their typical reliance on detergents in the early stages of membrane extraction may be problematic

Summary

Introduction

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. 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. A8-35 and the other classical APols traditionally require initial detergent extraction of the protein[31] This limitation has been overcome with the development of cycloalkane-modified APols (which contain cyclic rather than linear aliphatic groups) showing much greater efficiency at extracting proteins directly from the membrane than the common A8-35 APol[32]. SMALPs have their limitations (sensitivity to pH extremes and divalent cations), as such there is a continuous interest in developing new SMA-like polymers such as the acrylic acid and styrene polymers (AASTY)[42] which can be used to directly extract proteins from the membrane, but currently, their applicability to cryoEM has been limited to ~18 Å resolution. It has recently been demonstrated that A8-35 can be utilised following protein extraction with SMA44, an APol-like polymer combining the extraction capability of SMA with the applications of A8-35 would be highly advantageous

Methods

Results

Conclusion