Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Department of Biochemistry and Molecular Biophysics, Center for Science and Engineering of Living Systems (CSELS), Washington University in St. Louis, St. Louis, Missouri 63130, United States.
J Phys Chem B. 2021 Jun 24;125(24):6617-6628. doi: 10.1021/acs.jpcb.1c02370. Epub 2021 Jun 14.
Single-molecule Förster resonance energy transfer (FRET) is well suited for studying the kinetics of protein conformational changes, owing to its high sensitivity and ability to resolve individual subpopulations in heterogeneous systems. However, the most common approach employing two fluorophores can only monitor one distance at a time, and the use of three fluorophores for simultaneously monitoring multiple distances has largely been limited to equilibrium fluctuations. Here we show that three-color single-molecule FRET can be combined with rapid microfluidic mixing to investigate conformational changes in a protein from milliseconds to minutes. In combination with manual mixing, we extended the kinetics to 1 h, corresponding to a total range of 5 orders of magnitude in time. We studied the monomer-to-protomer conversion of the pore-forming toxin cytolysin A (ClyA), one of the largest protein conformational transitions known. Site-specific labeling of ClyA with three fluorophores enabled us to follow the kinetics of three intramolecular distances at the same time and revealed a previously undetected intermediate. The combination of three-color single-molecule FRET with rapid microfluidic mixing thus provides an approach for probing the mechanisms of complex biomolecular processes with high time resolution.
单分子Förster 共振能量转移（FRET）非常适合研究蛋白质构象变化的动力学，因为它具有高灵敏度和在异质体系中分辨单个亚群的能力。然而，最常用的采用两个荧光团的方法一次只能监测一个距离，而使用三个荧光团同时监测多个距离在很大程度上仅限于平衡波动。在这里，我们展示了三色单分子 FRET 可以与快速微流混合相结合，从毫秒到分钟研究蛋白质的构象变化。与手动混合相结合，我们将动力学扩展到 1 小时，对应于时间上 5 个数量级的总范围。我们研究了孔形成毒素细胞溶素 A（ClyA）的单体到原聚体的转化，这是已知的最大蛋白质构象转变之一。通过三个荧光团对 ClyA 进行位点特异性标记，使我们能够同时跟踪三个分子内距离的动力学，并揭示了以前未检测到的中间体。因此，三色单分子 FRET 与快速微流混合的结合为用高时间分辨率研究复杂生物分子过程的机制提供了一种方法。
