Kon Takahide, Shima Tomohiro, Sutoh Kazuo
Department of Life Sciences, University of Tokyo, Japan.
Methods Cell Biol. 2009;92:65-82. doi: 10.1016/S0091-679X(08)92005-7. Epub 2009 Nov 21.
Dyneins are microtubule-based motor complexes that power a wide variety of motile processes within eukaryotic cells, including the beating of cilia and flagella and intracellular trafficking along microtubules. Mechanistic studies on dynein have been hampered by their enormous size (molecular masses of 0.5-3MDa) and molecular complexity. However, the recent establishment of recombinant expression systems for cytoplasmic dynein, together with structural and functional analyses, has advanced our understanding of the molecular mechanisms of dynein motility. Here, we describe several protocols for protein engineering approaches to the dynein mechanism using a Dictyostelium discoideum expression system. We first describe the design and preparation of recombinant dynein suitable for mechanistic studies. We then discuss two distinct functional assays that take advantage of the recombinant dynein. One is for detection of dynein's conformational changes during the ATPase cycle. Another is an in vitro motility assay at multiple- and single-molecule levels for examination of the dynamic behavior of dynein moving on a microtubule.
动力蛋白是基于微管的马达复合体，为真核细胞内多种运动过程提供动力，包括纤毛和鞭毛的摆动以及沿微管的细胞内运输。对动力蛋白的机制研究因其巨大的尺寸（分子量为0.5 - 3MDa）和分子复杂性而受到阻碍。然而，最近细胞质动力蛋白重组表达系统的建立，以及结构和功能分析，增进了我们对动力蛋白运动分子机制的理解。在这里，我们描述了几种使用盘基网柄菌表达系统对动力蛋白机制进行蛋白质工程方法的方案。我们首先描述适合机制研究的重组动力蛋白的设计和制备。然后我们讨论利用重组动力蛋白的两种不同的功能测定法。一种用于检测动力蛋白在ATP酶循环期间的构象变化。另一种是在多分子和单分子水平上的体外运动测定法，用于检查动力蛋白在微管上移动的动态行为。
