A mutation in switch I alters the load-dependent kinetics of myosin Va.
A mutation in switch I alters the load-dependent kinetics of myosin Va.
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DOI：
10.1038/s41467-023-38535-0
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发表时间：
2023-05-30
影响因子：
16.6
通讯作者：
Debold, Edward P.
中科院分区：
文献类型：
--
作者：
Marang, Christopher;Scott, Brent;Chambers, James;Gunther, Laura K.;Yengo, Christopher M.;Debold, Edward P.
肌球蛋白 Va 是驱动细胞内囊泡运输的分子马达，由 ATP 的化学能转化为机械功提供动力。动力冲程和磷酸盐 (Pi) 释放的耦合是理解转导过程的关键，但该过程的关键细节仍不清楚。因此，我们在激光陷阱测定中确定了 Pi 升高对肌球蛋白 Va S1 (WT) 迷你整体产生力能力的影响。通过增加激光陷阱的刚度，我们确定了增加电阻负载对 Pi 诱导的肌动蛋白分离速率的影响，并使用贝尔近似量化了这种影响。我们观察到，在 30 mM Pi 存在的情况下，WT 肌球蛋白在较高的激光陷阱刚度下产生更高的力和更大的位移，但结合事件寿命急剧下降，这与 Pi 从活性位点释放之前的动力冲程最为一致。使用活性位点开关 I (S217A) 中具有突变的构建体重复这些实验，导致 Pi 诱导的脱离率的负载依赖性增加七倍，表明开关 I 的 S217A 区域可能有助于介导 Pi 重新结合的负载依赖性。肌球蛋白将化学能转化为机械功，但其机制仍不清楚。在这项工作中，作者表明，力的产生先于产品释放，并且活性位点的突变改变了产品释放的负载依赖性。
Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (Pi) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated Pi on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of Pi-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM Pi, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of Pi from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the Pi-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of Pi-rebinding. Myosin transduces chemical energy into mechanical work, but the mechanism remains unclear. In this work, the authors show that force-generation precedes product release and that a mutation in the active site alters the load dependence of product release.
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DOI：
10.1016/j.str.2010.01.014
发表时间：
2010-03-14
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Cecchini M;Alexeev Y;Karplus M
通讯作者：
Karplus M
影响因子：
5.5
作者：
COOKE, R;FRANKS, K;PATE, E
通讯作者：
PATE, E
影响因子：
2.7
作者：
Dupuis, DE;Guilford, WH;Warshaw, DM
通讯作者：
Warshaw, DM
影响因子：
3.4
作者：
Kad, Neil M.;Patlak, Joseph B.;Warshaw, David M.
通讯作者：
Warshaw, David M.
影响因子：
64.8
作者：
FINER, JT;SIMMONS, RM;SPUDICH, JA
通讯作者：
SPUDICH, JA