Use of stopped-flow fluorescence and labeled nucleotides to analyze the ATP turnover cycle of kinesins.
Use of stopped-flow fluorescence and labeled nucleotides to analyze the ATP turnover cycle of kinesins.
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DOI：
10.3791/52142
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发表时间：
2014-10-17
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影响因子：
--
通讯作者：
Friel CT
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文献类型：
--
作者：
Patel JT;Belsham HR;Rathbone AJ;Friel CT
微管相关运动蛋白的驱动蛋白超家族共享一个特征性的运动域，其既水解ATP又结合微管。驱动蛋白在ATP周转和微管相互作用两个超家族中都显示出差异。这些差异使特定的驱动蛋白适应各种功能，如货物运输、微管滑动、微管解聚和微管稳定。为了理解驱动蛋白的作用机制，重要的是要理解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周转周期，一种方法是利用荧光标记的核苷酸来可视化周期中的各个步骤。确定ATP周转周期中每个核苷酸转换的动力学允许鉴定完整周期的一个或多个限速步骤。对于驱动蛋白，重要的是要知道在没有微管的情况下的限速步骤，因为当驱动蛋白与微管相互作用时，该步骤通常被加速数千倍。然后将不存在微管的周期与存在微管的周期进行比较，以充分了解驱动蛋白的ATP周转周期。单个核苷酸转换的动力学通常太快而不能通过手动混合反应物来观察，特别是在微管存在的情况下。可以使用快速混合装置，例如停流荧光计，其允许在短至几毫秒的时间尺度上观察动力学，以监测这种转变。在这里，我们描述的协议，其中快速混合的试剂，通过停流与荧光标记的核苷酸一起使用解剖ATP周转周期的驱动蛋白。
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.
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