A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.
A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.
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DOI：
10.1016/s0076-6879(10)72008-5
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发表时间：
2010
影响因子：
--
通讯作者：
Gonzalez RL Jr
中科院分区：
文献类型：
--
作者：
Fei J;Wang J;Sternberg SH;MacDougall DD;Elvekrog MM;Pulukkunat DK;Englander MT;Gonzalez RL Jr
单分子荧光共振能量转移（smFRET）已成为越来越复杂的生化系统机制研究的有力工具。最近，我们和其他人已经成功地使用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。这些实验和类似的复杂细胞机器实验面临的一个重大挑战是，需要在既具有机械信息又对生物活性具有最小扰动的位置对生化系统进行特定和有效的荧光标记。在这里，我们描述了一个高度纯化，荧光标记的体外翻译系统的发展，我们已经成功地为蛋白质合成的smFRET研究设计。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。
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.