Membrane proteins represent ~ 30% of open reading frames in the human genome, ~ 70% of drug targets1 and yet are only 3% of reported structures in the PDB. Despite their prevalence in the cell and importance for ion transport and cell signalling, amongst other functions they remain challenging research targets due to problems of overexpression, extraction and stabilisation of their native structure2–5. Traditionally extraction and purification of a membrane protein involves the use of a detergent, from which the protein may then be transferred into other surfactants, be they detergents of different chemical composition, protein-based nanodiscs or amphipols6,7. Extraction of a membrane protein into a detergent micelle functions by disrupting the interaction between protein and its surrounding lipid molecules8. Detergent molecules recover the hydrophobic surface of a membrane protein but poorly mimic the lipid bilayer in terms of lateral pressure and thickness9 which has been shown to cause perturbations in the structure9,10. Moreover, the closely associated lipids which can be important for gating, regulation and stability, may be displaced by competition with the detergent11–14. In addition, detergent purification buffers must retain the detergent above its critical micelle concentration (CMC) in all downstream steps which may exacerbate lack of activity, dissociation of a protein complex, unnatural oligomerisation and loss of lipid cofactors, amongst other problems15–17. Detergent micelles in single-particle cryoEM lead to reduced contrast and increased noise18,19 and must be disassembled in native mass spectrometry (MS)20. Due to the importance of membrane proteins and the problems associated with detergents, there exist several alternative membrane mimetics developed to circumvent this, of which the predominant are protein-based nanodiscs21 and amphipathic polymers22.
Classical amphipols (APols) are short and flexible amphipathic polymers able to form complexes with membrane proteins and maintain the proteins in a water-soluble form22. They have been established for decades22,23 and are well-characterised in their applicability for stabilising membrane proteins. The prototypical APol A8-35 is a poly(acrylic acid) (PAA) polymer randomly modified with octylamine and isopropylamine side chains23, and many different functionalities have been tethered to the polymer for specific purposes24,25. In cryo-EM, APol A8-35 facilitated the first high-resolution single-particle structure of a membrane protein, that of TRPV126. Since then, the number of high-resolution cryoEM structures of membrane proteins using APols (mainly A8-35 and PMAL-C8)27 has increased28. Of those cryoEM structures deposited within the EMDB, the best resolution achieved using classical APols is 2.17 Å29. In addition, APols are amenable to native electrospray ionization (ESI)-MS30. However, A8-35 and the other classical APols traditionally require initial detergent extraction of the protein31. Other polymers are under development, such as the novel acrylic acid and styrene polymers (AASTY)32, but their applicability to cryoEM has been limited to ~ 18 Å resolution.
Conversely, the copolymerisation of styrene and maleic acid (SMA)33 heralded the advent of “native” nanodiscs containing a protein directly extracted from the membrane, with its endogenous lipids and without the requirement for conventional detergents34–39. The styrene maleic acid lipid particles (SMALPs) formed40 lend themselves to a plethora of biophysical techniques, including cryoEM41,42. However, SMALPs also have their limitations; they are more sensitive to pH extremes and divalent cations than PAA-derivative APols, making them incompatible for some activity assays37,43 while no MA-derived polymers have yet been successfully applied to native MS. Although 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.
Here we demonstrate that the properties of A8-35 and SMA can be combined through novel cycloalkane-modified APols with SMALP-like properties for direct extraction45. Using Escherichia coli AcrB, we demonstrate that these novel APol derivatives (henceforth distinguished as CyclAPols) are capable of solubilising the protein of interest directly from the membrane. The CyclAPols can be utilised at exceptionally low concentrations (0.1–0.5%) decreasing purification costs, and minimizing the risk of destabilisation due to high APol concentrations46,47. We present the first cryoEM structure of a protein in CyclAPols, at 3.2 Å resolution, demonstrating their applicability to high-resolution structure determination, making these APols an important new tool in the study of membrane proteins.