Single molecule fluorescence for membrane proteins
Single molecule fluorescence for membrane proteins
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DOI：
10.1016/j.ymeth.2018.05.024
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发表时间：
2018-09-01
期刊：
影响因子：
4.8
通讯作者：
Goddard, Alan D.
中科院分区：
文献类型：
--
作者：
Castell, Oliver K.;Dijkman, Patricia M.;Goddard, Alan D.
细胞膜是脂质和蛋白质的复杂环境。为了了解单个分子的行为，通常需要在体外系统中将它们作为纯化组分进行检查。在这里，我们详细介绍了液滴界面双层（DIBs）的创建和使用，当耦合到TIRF显微镜，可以揭示单个膜蛋白的时空和动力学信息。需要许多步骤，包括修饰蛋白质序列以能够掺入适当的荧光标记、表达和纯化膜蛋白以及随后的标记。在产生DIB后，蛋白质自发地掺入膜中，在膜中它们可以通过常规的单分子TIRF方法成像。使用这种策略，结合逐步光漂白，FRET和/或单粒子跟踪，可以确定一系列参数，如寡聚状态和动态信息。我们讨论了这个系统的优点和局限性，并为这些方法的成功实施提供指导。
The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and/or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches.