Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry
Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry
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DOI：
10.1073/pnas.96.2.465
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发表时间：
1999-01-19
影响因子：
11.1
通讯作者：
White, HD
中科院分区：
文献类型：
--
作者：
Walker, M;Zhang, XZ;White, HD
用停流荧光和ms时间分辨电镜技术研究了在无核苷酸存在下肌球蛋白亚片段1（myosin subfragment-1，S1）与肌动蛋白的结合机制。这表明在碰撞复合物之后和稳定的“僵硬”复合物形成之前存在瞬时中间体。瞬时中间体在混合后2-15 ms占主导地位，而僵硬复合物在>50 ms时占主导地位。混合后10 ms冷冻的acto-S1的电子显微镜显示无序结合。在50 ms或更长时间冷冻的Acto-S1显示出僵直的“箭头”外观特征。对瞬时中间体紊乱的最可能的解释是，结合是通过蛋白质表面上的一个或多个柔性环。从无序到有序结合的转变可能是肌肉中产生力的步骤的一部分。
The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy, The fluorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable "rigor" complex, The transient intermediate predominates 2-15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the "arrow-head" appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more flexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.