Clobes Amy M, Guilford William H
Department of Biomedical Engineering, University of Virginia, Box 800759, Charlottesville, VA, 22908, USA.
J Muscle Res Cell Motil. 2014 Apr;35(2):143-52. doi: 10.1007/s10974-014-9375-z. Epub 2014 Feb 6.
Myosin's actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state-the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.
肌球蛋白的肌动蛋白结合环（环2）所带电荷与其在肌动蛋白上的结合位点电荷相反，并且在初始结合步骤中，该环被认为在两个分子之间的离子相互作用中发挥重要作用。然而，尚未确定环2在肌动蛋白 - 肌球蛋白结合过程中的后续作用。我们使用光镊来测量垂直于细丝轴加载时肌动蛋白与僵直重酶解肌球蛋白之间的键形成和键断裂。我们研究了在低离子强度和生理离子强度下，环2完整或经蛋白酶切割的重酶解肌球蛋白。在此我们表明，完整环2的存在使肌动球蛋白键能够快速形成，并且它们以短暂结合状态形成。增加拉伸负荷会导致向长寿命状态的转变——这是一种捕获键的显著行为。当环2被切割时，捕获键行为消失，仅留下长寿命状态。这些数据表明，除了其在定位肌动蛋白上结合位点的作用外，环2还是长寿命肌动球蛋白复合物的力依赖性抑制剂。这对于在低力情况下降低工作比和提高肌动球蛋白的缩短速度可能很重要。
