一种用于蛋白质合成单分子研究的高度纯化的、荧光标记的体外翻译系统。
A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.
作者信息
Fei Jingyi, Wang Jiangning, Sternberg Samuel H, MacDougall Daniel D, Elvekrog Margaret M, Pulukkunat Dileep K, Englander Michael T, Gonzalez Ruben L
机构信息
Department of Chemistry, Columbia University, New York, NY, USA.
出版信息
Methods Enzymol. 2010;472:221-59. doi: 10.1016/S0076-6879(10)72008-5.
Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful tool for mechanistic investigations of increasingly complex biochemical systems. Recently, we and others have successfully used smFRET to directly investigate the role of structural dynamics in the function and regulation of the cellular protein synthesis machinery. A significant challenge to these experiments, and to analogous experiments in similarly complex cellular machineries, is the need for specific and efficient fluorescent labeling of the biochemical system at locations that are both mechanistically informative and minimally perturbative to the biological activity. Here, we describe the development of a highly purified, fluorescently labeled in vitro translation system that we have successfully designed for smFRET studies of protein synthesis. The general approaches we outline should be amenable to single-molecule fluorescence studies of other complex biochemical systems.
单分子荧光共振能量转移（smFRET）已成为研究日益复杂的生化系统机制的有力工具。最近，我们和其他人已成功使用smFRET直接研究结构动力学在细胞蛋白质合成机制的功能和调节中的作用。这些实验以及类似复杂细胞机制中的类似实验面临的一个重大挑战是，需要在对生物活性具有最小干扰且在机制上具有信息性的位置对生化系统进行特异性和高效的荧光标记。在这里，我们描述了一种高度纯化的、荧光标记的体外翻译系统的开发，我们已成功设计该系统用于蛋白质合成的smFRET研究。我们概述的一般方法应该适用于其他复杂生化系统的单分子荧光研究。
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