Confocal single-molecule FRET for protein conformational dynamics.
Confocal single-molecule FRET for protein conformational dynamics.
复制标题
DOI：
10.1007/978-1-62703-658-0_3
复制
发表时间：
2014
影响因子：
--
通讯作者：
Yan-Wen Tan;J. A. Hanson;J. Chu;Haw Yang
中科院分区：
文献类型：
--
作者：
Yan-Wen Tan;J. A. Hanson;J. Chu;Haw Yang
单分子F受体型共振能量转移（smFRET）是一种能够跟踪单个蛋白质分子构象运动的独特技术。对单个蛋白质的直接观察提供了丰富的信息，这些信息在系综测量中会被冲走，从而为通过动力学建立蛋白质结构-功能关系开辟了新的途径。然而，通过smFRET检索生物分子运动的动力学信息需要仔细的实验设计和严格的单分子统计处理。在这里，我们描述了一个smFRET实验的基本步骤，包括样品制备显微镜，单分子FRET检测的关键部分的建设，和一个强大的方法，光子的光子数据分析。
Single-molecule Fӧrster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis.