Department of Pharmacology, Yale University, New Haven, CT, USA.
Department of Cell Biology, Yale University, New Haven, CT, USA.
Nat Nanotechnol. 2024 Jan;19(1):85-94. doi: 10.1038/s41565-023-01547-4. Epub 2023 Nov 27.
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.
天然细胞膜中膜蛋白的寡聚化组织是其功能的关键调节剂。对寡聚体组装及其在不同条件下如何变化的高分辨率定量测量对于理解膜蛋白生物学是不可或缺的。我们报告了 Native-nanoBleach，这是一种基于全内反射荧光显微镜的单分子光漂白分步分析技术，可直接从天然膜中以约 10nm 的有效空间分辨率确定膜蛋白的寡聚分布。我们通过使用两亲性共聚物将靶标膜蛋白捕获在其近天然膜环境中的天然纳米盘中来实现这一点。我们应用 Native-nanoBleach 来定量测量结构和功能多样化的膜蛋白的寡聚状态，包括受体酪氨酸激酶（TrkA）和小 GTP 酶（KRas），分别在生长因子结合和致癌突变下。我们的数据表明，Native-nanoBleach 提供了一种敏感的单分子平台，可在生理和临床相关条件下定量测量天然膜中膜蛋白的寡聚分布。
