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The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to understanding membrane protein biology. We report Native-nanoBleach, a total internal reflection fluorescence microscopy-based single-molecule photobleaching step analysis technique to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ~10 nm. We achieved this by capturing target membrane proteins in native nanodiscs with their proximal native membrane environment using amphipathic copolymers. We applied Native-nanoBleach to quantify the oligomerization status of structurally and functionally diverse membrane proteins, including a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under growth-factor binding and oncogenic mutations, respectively. Our data suggest that Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes under physiologically and clinically relevant conditions. © 2023. The Author(s), under exclusive licence to Springer Nature Limited.

Citation

Gerard Walker, Caroline Brown, Xiangyu Ge, Shailesh Kumar, Mandar D Muzumdar, Kallol Gupta, Moitrayee Bhattacharyya. Oligomeric organization of membrane proteins from native membranes at nanoscale spatial and single-molecule resolution. Nature nanotechnology. 2024 Jan;19(1):85-94

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PMID: 38012273

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