Walker Gerard, Brown Caroline, Ge Xiangyu, Kumar Shailesh, Muzumdar Mandar D, Gupta Kallol, Bhattacharyya Moitrayee
bioRxiv. 2023 Feb 21:2023.02.19.529138. doi: 10.1101/2023.02.19.529138.
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.
膜蛋白在天然细胞膜中的寡聚组织是其功能的关键调节因子。对寡聚体组装体及其在不同条件下如何变化进行高分辨率定量测量，对于理解膜蛋白生物学不可或缺。我们报告了一种单分子成像技术（天然纳米漂白法），可在约10纳米的有效空间分辨率下直接从天然膜中确定膜蛋白的寡聚分布。我们通过使用两亲性共聚物将目标膜蛋白捕获在“天然纳米盘”及其近端天然膜环境中来实现这一点。我们使用具有明确化学计量比的结构和功能多样的膜蛋白建立了该方法。然后，我们分别应用天然纳米漂白法来量化受体酪氨酸激酶（TrkA）和小GTP酶（KRas）在生长因子结合或致癌突变条件下的寡聚化状态。天然纳米漂白法提供了一个灵敏的单分子平台，以前所未有的空间分辨率量化天然膜中膜蛋白的寡聚分布。
