Abstract
Amphiphilic copolymers capable of extracting membrane proteins directly from cellular membranes into “native nanodiscs” offer a simplified approach for preparing membrane proteins in lipid nanodiscs compared to approaches that rely on detergent. Copolymer amphiphilicity, length, and composition influence their performance, in addition to the protein itself and the purification conditions used. Here, we report a copolymer composed of methacrylic acid and styrene, which we term MAASTY, leveraging the inherent monomer reactivity ratios to create an anionic copolymer with a statistical distribution of monomers. We show that MAASTY can be used for high-resolution structural determination of a human membrane protein by single particle cryo-electron microscopy, preserving endogeno…
Abstract
Amphiphilic copolymers capable of extracting membrane proteins directly from cellular membranes into “native nanodiscs” offer a simplified approach for preparing membrane proteins in lipid nanodiscs compared to approaches that rely on detergent. Copolymer amphiphilicity, length, and composition influence their performance, in addition to the protein itself and the purification conditions used. Here, we report a copolymer composed of methacrylic acid and styrene, which we term MAASTY, leveraging the inherent monomer reactivity ratios to create an anionic copolymer with a statistical distribution of monomers. We show that MAASTY can be used for high-resolution structural determination of a human membrane protein by single particle cryo-electron microscopy, preserving endogenous lipids including cholesterol and exhibiting an enrichment of phosphatidylinositol. Moreover, MAASTY copolymers effectively solubilize a broad range of lipid species and a wide range of different, eukaryotic membrane proteins from mammalian cells. We find that MAASTY copolymers are promising as effective solubilizers of membrane proteins and offer a chemical platform for structural and functional characterization of membrane proteins in native nanodiscs.
Data availability
The reconstructed maps are available from the Electron Microscopy Data Bank (EMDB) under accession codes EMD-52611 and EMD-55036. The atomic coordinates based on the reconstructions have been deposited in the Protein Data Bank (PDB) under accession codes PDB 9I3R and 9SMK. The raw cryo-EM movies and particles stacks have been deposited on the Electron Microscopy Public Image Archive (EMPIAR) and are available under accession codes EMPIAR-12546 and EMPIAR-13000. Plasmids and other data that support the findings of this study are available from the corresponding authors. Source data for all main figures is available from Figshare [https://doi.org/10.6084/m9.figshare.30213958].
Code availability
The data analysis scripts for plotting fluorescent size exclusion chromatography traces in this study were implemented using Python 3.10.6. All scripts and computational workflows necessary to reproduce the results are available upon request from the corresponding authors.
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Acknowledgements
The authors thank senior research scientists Tillmann Hanns Pape, Nicholas Heelund Sofos, and Michael James Johnson for their support for cryo-EM experiments at the Core Facility for Integrated Microscopy at the University of Copenhagen. The authors thank members of the Autzen Group for data discussion. hTRPM4, rSERCA, and hKCNK18 were expressed in a modified version of a BacMam vector gifted from Eric Gouaux (Addgene plasmid # 160680). The plasmid for expressing cASIC1 was gifted from Eric Gouaux. Subsets of the figures were created with BioRender.com. H.E.A. acknowledges the Novo Nordisk Foundation (NNF20OC0060692), the Carlsberg Foundation (CF20-0533), and Independent Research Fund Denmark (1131-00023B) for support. A.A.A.A. acknowledges Independent Research Fund Denmark (0171-00081B) for support. J.R.B. acknowledges the Royal Society for the support through the University Research Fellowship grant (URF\R1\211567).
Author information
Authors and Affiliations
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, N, DK-2200, Denmark
Ciara F. Pugh, Lukas P. Feilen, Casper de Lichtenberg & Henriette E. Autzen 1.
Department of Biology, University of Oxford, Oxford, OX1 3RB, UK
Dušan Živković & Jani R. Bolla 1.
Department of Health Technology, Technical University of Denmark, Kgs. Lyngby, DK-2800, Denmark
Kaia F. Præstegaard, Charalampos Sideris, Neil J. Borthwick & Anton A. A. Autzen
Authors
- Ciara F. Pugh
- Lukas P. Feilen
- Dušan Živković
- Kaia F. Præstegaard
- Charalampos Sideris
- Neil J. Borthwick
- Casper de Lichtenberg
- Jani R. Bolla
- Anton A. A. Autzen
- Henriette E. Autzen
Contributions
H.E.A. and A.A.A.A. conceived the project. C.F.P., H.E.A., and A.A.A.A. designed the experimental procedures. A.A.A.A., K.F.P., C.S., and N.J.B. carried out copolymer synthesis. N.J.B. carried out NMR. C.F.P. carried out expression of all membrane proteins, FSEC screening of membrane proteins and fluorescent lipid nanodiscs, purification and vitrification of hTRPM4 in MAASTY nanodiscs, and preparation of hTRPM4 for lipidomics and MP analysis. L.P.F. purified hTRPM4 in MSP2N2 nanodiscs, and C.d.L vitrified the sample for cryo-EM. C.F.P. and L.P.F. performed image acquisition. C.F.P. and H.E.A. performed single particle analysis. C.F.P. built atomic models. K.F.P. carried out calcium-binding experiments and pH sensitivity. A.A.A.A. did MALDI-TOF analysis of hTRPM4 in MAASTY. D.Ž. and J.R.B. carried out lipidomics analysis and MP. C.F.P., H.E.A. and A.A.A.A wrote the manuscript. C.F.P., H.E.A., and A.A.A.A. prepared figures. All authors read and reviewed the manuscript.
Corresponding authors
Correspondence to Anton A. A. Autzen or Henriette E. Autzen.
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Competing interests
A.A.A.A., N.J.B., and H.E.A. have submitted a patent application (current status: pending) to the International Searching Authority (ISA) pertaining to solubilizing membrane proteins and lipids in nanodiscs composed of methacrylic acid-co-styrene copolymers (application number WO2024153743A1). All other authors declare no competing interests.
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Pugh, C.F., Feilen, L.P., Živković, D. et al. MAASTY: a (dis)ordered copolymer for structural determination of human membrane proteins in native nanodiscs. Nat Commun (2025). https://doi.org/10.1038/s41467-025-66208-7
Received: 13 August 2024
Accepted: 31 October 2025
Published: 10 December 2025
DOI: https://doi.org/10.1038/s41467-025-66208-7