Patel Jennifer T, Belsham Hannah R, Rathbone Alexandra J, Friel Claire T
School of Life Sciences, University of Nottingham.
School of Life Sciences, University of Nottingham;
J Vis Exp. 2014 Oct 17(92):e52142. doi: 10.3791/52142.
The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin's ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.
微管相关运动蛋白的驱动蛋白超家族共享一个既水解ATP又结合微管的特征性运动结构域。驱动蛋白在整个超家族中，在ATP周转和微管相互作用方面都存在差异。这些差异使特定的驱动蛋白适用于各种功能，如货物运输、微管滑动、微管解聚和微管稳定。要了解驱动蛋白的作用机制，重要的是要了解ATP周转的化学循环如何与微管相互作用的机械循环相耦合。为了剖析ATP周转循环，一种方法是利用荧光标记的核苷酸来可视化循环中的各个步骤。确定ATP周转循环中每个核苷酸转变的动力学，可以识别整个循环的限速步骤。对于驱动蛋白来说，了解在没有微管时的限速步骤很重要，因为当驱动蛋白与微管相互作用时，这个步骤通常会加速几千倍。然后将没有微管时的循环与有微管时的循环进行比较，以全面了解驱动蛋白的ATP周转循环情况。单个核苷酸转变的动力学通常太快，无法通过手动混合反应物来观察，特别是在有微管存在的情况下。一种快速混合装置，如停流荧光计，它可以在短至几毫秒的时间尺度上观察动力学，可用于监测这种转变。在这里，我们描述了一些实验方案，其中通过停流快速混合试剂，并结合荧光标记的核苷酸来剖析驱动蛋白的ATP周转循环。
