DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides
Membrane scaffold protein-based nanodiscs have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in nanodiscs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of nanodiscs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional nanodisc technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we create a class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free nanodiscs with high efficiency. Nanodiscs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of our approach, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.
Authors
- Ilya Levental (ORCID: https://orcid.org/0000-0002-1206-9545)
- Baron Chanda (ORCID: https://orcid.org/0000-0003-4954-7034)
- Huan Bao (ORCID: https://orcid.org/0000-0002-4301-4627)
- Lukas K. Tamm
- Vinay Idikuda (ORCID: https://orcid.org/0000-0001-6324-5839)
- Ahmad Jomaa (ORCID: https://orcid.org/0000-0002-5543-7942)
- Sara Zdancewicz (ORCID: https://orcid.org/0000-0002-1987-6817)
- Jing Wang (ORCID: https://orcid.org/0000-0003-3378-2099)
- Alex J.B. Kreutzberger (ORCID: https://orcid.org/0000-0002-9774-115X)
- Luis M. Real Hernandez
- Volker Kiessling
- W.H. Ludlam (ORCID: https://orcid.org/0000-0002-2701-8791)
- Kirill A. Martemyanov (ORCID: https://orcid.org/0000-0002-9925-7599)
- Shanwen Zhang (ORCID: https://orcid.org/0000-0002-4534-5358)
- Qian Ren
- H. S. Chang
- Jeehae Shin (ORCID: https://orcid.org/0000-0003-3127-3450)
Institutions
- Scripps Research Institute (US)
- Washington University in St. Louis (US)
- New York Structural Biology Center (US)
- Scripps (United States) (US)
- The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology
- University of Virginia (US)
- Hankuk University of Foreign Studies (KR)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-08-26
- DOI
- https://doi.org/10.1038/s41467-025-63275-8
- Citations
- 4
- Primary Topic
- Advanced biosensing and bioanalysis techniques
- Type
- article
- Field-Weighted Citation Impact
- 3.22
Funders
- Institute of Clinical and Translational Sciences
- Alvin J. Siteman Cancer Center
- Georgia Clinical and Translational Science Alliance
- Hankuk University of Foreign Studies
- National Institutes of Health
- National Institute on Aging
- National Cancer Institute
- National Institute of General Medical Sciences
- National Center for Advancing Translational Sciences
- Oak Ridge National Laboratory
- Common Fund