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bioRxiv · 10.1101/2023.02.19.529138

Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution

Walker, G.·Brown, C.·Ge, X.·Kumar, S.·Muzumdar, M. D.·Gupta, K.·Bhattacharyya, M.
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Abstract

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 the understanding of membrane protein biology. We report a single-molecule imaging technique (Native-nanoBleach) 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 established this method using structurally and functionally diverse membrane proteins with well-established stoichiometries. We then applied Native-nanoBleach to quantify the oligomerization status of a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under conditions of growth-factor binding or oncogenic mutations, respectively. Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes at an unprecedented spatial resolution.

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Walker, G., Brown, C., Ge, X., Kumar, S., Muzumdar, M. D., Gupta, K., Bhattacharyya, M.. 2023-02-21. Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution. https://doi.org/10.1101/2023.02.19.529138

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