Friel Claire T, Bagshaw Clive R, Howard Jonathon
School of Biomedical Sciences, University of Nottingham, Nottingham, UK.
Methods Mol Biol. 2011;777:177-92. doi: 10.1007/978-1-61779-252-6_13.
Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5'-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubule interaction. In this chapter, a number of methods are outlined by which the ATP turnover cycle of a kinesin can be analysed with a particular focus on the use of fluorescently labelled ATP and ADP analogues as a means of isolating individual steps in the cycle. By analysing the ATP turnover cycle of a kinesin, both in solution and in the presence of microtubules, the change in nucleotide state triggered upon microtubule binding can be determined. This provides information vital to understanding the coupling of the chemical and mechanical cycles that is integral to the action of members of the kinesin superfamily.
驱动蛋白超家族的蛋白质共享一个保守的运动结构域，该结构域既能水解腺苷-5'-三磷酸（ATP），又能结合微管。为了确定驱动蛋白的作用机制，有必要将ATP周转的化学循环与微管相互作用的力学联系起来。在本章中，概述了一些方法，通过这些方法可以分析驱动蛋白的ATP周转循环，特别关注使用荧光标记的ATP和ADP类似物作为分离循环中各个步骤的手段。通过分析溶液中和存在微管时驱动蛋白的ATP周转循环，可以确定微管结合时触发的核苷酸状态变化。这为理解化学循环和机械循环的耦合提供了至关重要的信息，而这种耦合是驱动蛋白超家族成员作用所不可或缺的。
