Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
PLoS Biol. 2011 Apr;9(4):e1001031. doi: 10.1371/journal.pbio.1001031. Epub 2011 Apr 12.
Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.
肌球蛋白是一种 ATP 驱动的线性分子马达，作为细胞力发生器、转运器和力传感器发挥作用。这些功能是由大规模核苷酸依赖性构象变化驱动的，称为“冲程”；“力冲程”是指当肌球蛋白轻链结合“颈部”域结合在肌动蛋白上时相对于“头部”域摆动的力产生摆动；“恢复冲程”是使肌球蛋白脱离肌动蛋白时进行预位或“上膛”的必要初始运动。肌球蛋白 Va 是一种进行性二聚体，沿着肌动蛋白沿“手对手”机制单向步移，其中尾随的头部脱离并向前步移约 72nm。尽管尾部头部围绕两个颈部之间的自由关节进行大的旋转布朗运动，但单向步移得以实现，部分原因是附着头部的力冲程向前移动关节。然而，仅有力冲程不能完全解释优先向前结合位点，因为脱离头部的取向和角度稳定性由恢复冲程的性质决定，决定了肌动蛋白结合位点的可及性。在这里，我们使用一种新颖的、瞬态笼式 ATP 控制系统直接观察肌球蛋白 Va 的恢复冲程动力学和波动，该系统通过不同强度的逐步 UV 脉冲序列维持恒定的 ATP 水平。我们将单体肌球蛋白 Va 的颈部固定在表面上，并观察到特定地附着在头部的珠子的实时运动。ATP 诱导颈部短暂摆动到恢复后冲程构象，在该构象中它保持约 40s，直到 ATP 水解产物被释放。角度分布表明，恢复后冲程构象通过≥5 k(B)T 的能量稳定。恢复后冲程构象的高动力学和高能量稳定性有利于脱离头部优先结合 72nm 远处的前位。因此，恢复冲程有助于肌球蛋白 Va 的单向步移。
