The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro
The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro
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DOI：
10.1074/jbc.m114.564740
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发表时间：
2014-07-25
影响因子：
4.8
通讯作者：
Cremo, Christine R.
中科院分区：
文献类型：
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作者：
Haldeman, Brian D.;Brizendine, Richard K.;Cremo, Christine R.
肌动蛋白-肌球蛋白相互作用的研究，使用可溶性肌球蛋白片段，但鲜为人知的是肌球蛋白丝结构的机械化学的影响。我们稳定非磷酸化平滑肌肌球蛋白（SMM）和磷酸化平滑肌肌球蛋白（pSMM）对ATP诱导的解聚使用交联剂和附加荧光罗丹明（XL-Rh-SMM）的细丝。电子显微照片显示，这些侧极性长丝与未改性的长丝非常相似。它们长约0.63 μ m，含有约176个分子。ATP诱导的解离和ADP释放肌动球蛋白的细丝和S1头的速率常数是相似的。SMM和XL-Rh-SMM的肌动蛋白激活的ATP酶受到类似的调节。XL-Rh-pSMM细丝在与PEG刷表面结合的F-肌动蛋白上向前移动。ATP依赖的细丝速度是类似的溶液ATP酶在高[肌动蛋白]，这表明这两个过程是有限的相同的动力学步骤（弱到强的过渡），因此是附着限制。这与肌动蛋白在肌球蛋白单体上滑动不同，肌动蛋白在肌球蛋白单体上滑动主要是限制性的。将细丝数据拟合到附件限制模型显示，大约一半的股骨头可用于移动细丝，与侧极结构一致。我们建议低刚度亚片段2（S2）域保持不受阻碍的细丝运动在我们的测定。由于S2屈曲，肌动蛋白结合的负位移股骨头将产生最小的阻力。考虑到ADP的释放速率、速度和S2的长度，这些头部将在松弛被吸收到向后移位的高刚度位置之前与肌动蛋白分离。该机制解释了在生理[ATP]下缺乏抑制限制动力学。这些发现解决了如何在组件的电机非线性弹性导致有效的集体力的产生。
Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are similar to 0.63 mu m long and contain similar to 176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation.