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

View ORCID ProfileGerard Walker, Caroline Brown, Xiangyu Ge, Shailesh Kumar, Mandar D. Muzumdar, Kallol Gupta, View ORCID ProfileMoitrayee Bhattacharyya
doi: https://doi.org/10.1101/2023.02.19.529138
Gerard Walker
1Department of Pharmacology, Yale University, New Haven, USA
2Department of Cell Biology, Yale University, New Haven, USA
3Nanobiology Institute, Yale University, New Haven, USA
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  • ORCID record for Gerard Walker
Caroline Brown
2Department of Cell Biology, Yale University, New Haven, USA
3Nanobiology Institute, Yale University, New Haven, USA
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Xiangyu Ge
4Department of Genetics, Yale University, New Haven, USA
5Yale Cancer Biology Institute, Yale University, New Haven, USA
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Shailesh Kumar
1Department of Pharmacology, Yale University, New Haven, USA
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Mandar D. Muzumdar
4Department of Genetics, Yale University, New Haven, USA
5Yale Cancer Biology Institute, Yale University, New Haven, USA
6Department of Internal Medicine, Yale University, New Haven, USA
7Yale Cancer Center, New Haven, USA
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Kallol Gupta
2Department of Cell Biology, Yale University, New Haven, USA
3Nanobiology Institute, Yale University, New Haven, USA
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Moitrayee Bhattacharyya
1Department of Pharmacology, Yale University, New Haven, USA
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  • ORCID record for Moitrayee Bhattacharyya
  • For correspondence: moitrayee.bhattacharyya@yale.edu
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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.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 21, 2023.
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Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution
Gerard Walker, Caroline Brown, Xiangyu Ge, Shailesh Kumar, Mandar D. Muzumdar, Kallol Gupta, Moitrayee Bhattacharyya
bioRxiv 2023.02.19.529138; doi: https://doi.org/10.1101/2023.02.19.529138
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Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution
Gerard Walker, Caroline Brown, Xiangyu Ge, Shailesh Kumar, Mandar D. Muzumdar, Kallol Gupta, Moitrayee Bhattacharyya
bioRxiv 2023.02.19.529138; doi: https://doi.org/10.1101/2023.02.19.529138

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