Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (P_i) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining P_i in its active site, by either elevating P_i in solution or by introducing a mutation in myosin's active site (S217A) to slow P_i-release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s⁻¹) and without a detectable delay, both in the absence and presence of 30 mM P_i. The elevated levels of P_i did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P_i rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower P_i release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P_i still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of P_i from the active site.

中文翻译：

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肌球蛋白的动力冲程发生在磷酸盐从活性部位释放之前

肌球蛋白是一个运动蛋白家族，负责各种形式的细胞运动，包括肌肉收缩和囊泡运输。肌球蛋白最基本的方面是它能够以力和/或运动的形式将来自 ATP 水解的化学能转化为机械功。转导过程的一个关键未解决问题是产生力的动力冲程相对于从活性部位释放磷酸盐 (P _i ) 的时间。我们检查了单头肌球蛋白Va的在单个分子激光陷阱测定以产生powerstroke同时保持P上的能力_我在其活性位点，通过任一升降P_我在溶液中或通过在肌球蛋白的活性位点（S217A）中引入突变，以慢P _i- 从活动站点释放。在与肌动蛋白丝结合后，WT 肌球蛋白在 30 mM P _i不存在和存在的情况下都迅速（≥500 s ^-1）并且没有可检测到的延迟产生功率斯托克。然而，升高的 P _i水平确实影响了事件寿命，消除了最长的 25% 的结合事件，证实了 P _i反弹到肌球蛋白的活性位点并加速了脱离。尽管 P _i释放速率较慢，但 S217A 构建体在与肌动蛋白结合后也产生了大小和速率相似的动力冲程。这些发现提供了直接证据，即肌球蛋白 Va 产生 P _i的动力冲程仍然在它的活动现场。因此，这些发现与动力冲程发生在 P _i从活动部位释放之前的模型最为一致。