Durham Ryan J, Latham Danielle R, Sanabria Hugo, Jayaraman Vasanthi
University of Texas Health Science Center at Houston, Houston, Texas.
Clemson University, Clemson, South Carolina.
Biophys J. 2020 Nov 17;119(10):1929-1936. doi: 10.1016/j.bpj.2020.10.009. Epub 2020 Oct 20.
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.
单分子荧光共振能量转移（smFRET）是研究生物大分子结构动力学的强大技术。smFRET通过基于冷冻电子显微镜、X射线晶体学和其他方法获得的静态结构，揭示蛋白质的构象景观和动态变化。将smFRET与静态结构相结合，可以直接关联动态构象与功能。在这里，我们讨论了不同的实验装置、荧光检测方案和数据分析策略，这些策略能够在不同时间尺度上研究谷氨酸信号传导的结构动力学。我们通过突出一系列广泛问题的研究来说明smFRET的多功能性，这些问题包括激活和运输机制、内在无序片段的作用以及生物系统中负责谷氨酸信号传导的亚基之间的变构和协同作用。
