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

Abstract

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.

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids

“…A recent study has reported that phospholipids were present in samples of Acrb-solubilized and purified in CyclAPols . However, the lipid analysis, performed by thin layer chromatography, did not permit to accurately identify and quantify lipid species.…”

“…Knowing that extraction efficiencies can vary between prokaryotic and eukaryotic MPs, additional work would be needed to confirm this property. The CyclAPols (now supplied by Cube Biotech) have recently been validated as appropriate amphipathic environments for determining high-resolution structures of MPs by cryo-EM, which bodes well for their usefulness. One may add that APol A8-35 can be chemically modified with different types of functionalities, such as affinity tags − or fluorophores, without affecting its MP-stabilizing properties.…”

Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

“…Mimicking this property of SMA eliminates the requirement for classical detergent mediated extraction and subsequent APol exchange that can, in some cases, result in MP destabilisation. A recent preprint reported the use of CyclAPols in the solubiliation of the model bacterial membrane transporter AcrB which subsequently yielded a 3.2 Å Cryo-EM structure [ 92 ].…”

Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

“…It is worth to be pointed out that Marconnet et al have recently revealed a novel cryo-EM compatible polymers, cycloalkane-modified amphipols (CyclAPols), which can provide a native membrane protein extraction but could not retain the well-organized native lipids plug within transmembrane (TM) domains. 22,23 (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…It is worth to be pointed out that Marconnet et al have recently revealed a novel cryo-EM compatible polymers, cycloalkane-modified amphipols (CyclAPols), which can provide a native membrane protein extraction but could not retain the well-organized native lipids plug within transmembrane (TM) domains. 22,23 Among the membrane-active polymers, the commercial SMA2000 with defined ratio 2: 1 of hydrophobic: hydrophilic domains has exhibited as the most effective solubilizer for several membrane protein model systems. 7,9,12,24 To overcome the limitations of SMA2000 and retain a good extraction efficiency, our novel polymers were produced through individual grafting of or 100%, herein, "x" values were varied to explore how such contents and structures of the amines influence membrane solubilization, purification, and stability.…”