Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
Songshan Lake Materials Laboratory, Dongguan, Guangdong 523808, China.
Nano Lett. 2021 Jan 13;21(1):485-491. doi: 10.1021/acs.nanolett.0c03941. Epub 2020 Dec 5.
Characterization of biomolecular dynamics at cellular membranes lags far behind that in solutions because of challenges to measure transmembrane trafficking with subnanometer precision. Herein, by introducing nonfluorescent quenchers into extracellular environment of live cells, we adopted Förster resonance energy transfer from one donor to multiple quenchers to measure positional changes of biomolecules in plasma membranes. We demonstrated the method by monitoring flip-flops of individual lipids and by capturing transient states of the host defense peptide LL-37 in plasma membranes. The method was also applied to investigate the interaction of the necroptosis-associated protein MLKL with plasma membranes, showing a few distinct depths of MLKL insertion. Our method is especially powerful to quantitate the dynamics of proteins at the cytosolic leaflets of plasma membranes which are usually not accessible by conventional techniques. The method will find wide applications in the systematic analysis of fundamental cellular processes at plasma membranes.
由于难以实现亚纳米精度的跨膜转运测量，细胞膜上生物分子动力学的特征描述远远落后于溶液中的情况。在此，我们通过将非荧光猝灭剂引入活细胞的细胞外环境，采用从一个供体到多个猝灭剂的Förster 共振能量转移，来测量质膜中生物分子的位置变化。我们通过监测单个脂质的翻转以及捕获质膜中宿主防御肽 LL-37 的瞬态状态来证明该方法。该方法还应用于研究与坏死相关的蛋白 MLKL 与质膜的相互作用，显示出 MLKL 插入的几个不同深度。我们的方法特别适用于定量质膜胞质小叶中蛋白质的动力学，而这些蛋白质通常无法通过传统技术进行检测。该方法将在系统分析质膜上基本细胞过程中得到广泛应用。
