Single kinesin molecules studied with a molecular force clamp
Single kinesin molecules studied with a molecular force clamp
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DOI：
10.1038/22146
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发表时间：
1999-07-08
期刊：
影响因子：
64.8
通讯作者：
Block, SM
中科院分区：
文献类型：
--
作者：
Visscher, K;Schnitzer, MJ;Block, SM
Kinesin是一种双头的、由ATP驱动的马达蛋白，它以8 nm的离散步长沿着微管连续移动，可能是通过按顺序(1-4)交替推进其每个头部。关于储存在ATP中的化学能是如何与机械位移耦合的分子细节仍不清楚。为了阐明这一问题，基于反馈驱动的光学陷阱能够维持单个运动蛋白马达的恒定负载，构建了一个力钳(5)。该仪器提供了前所未有的分子运动分辨率，并允许在受控外部载荷下进行机械力化学研究。对不同的ATP浓度和负荷下的动蛋白运动记录的分析揭示了几个新的特征。首先，动蛋白步进似乎在很大的力范围内与ATP的水解紧密耦合，每8纳米的机械推进只有一次水解。第二，动蛋白失速力依赖于ATP浓度。第三，载荷的增加降低了最大速度，但也提高了表观米氏常数。因此，动蛋白循环包含至少一个影响ATP分子结合并随后进行水解的速率的负载依赖的转变，很可能至少存在另一个影响周转次数的负载依赖的速率。综上所述，这些发现将有必要修改我们对运动蛋白如何发挥作用的理解。
Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence(1-4). Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors(5). The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis, It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.