The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes.
The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes.
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发表时间：
1995
影响因子：
3.4
通讯作者：
David D. Thomas;Sampath Ramachandran;O. Roopnarine;David W. Hayden;E. Ostap
中科院分区：
文献类型：
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作者：
David D. Thomas;Sampath Ramachandran;O. Roopnarine;David W. Hayden;E. Ostap
我们提出了一个在肌肉中产生力量的分子机制，主要是基于对收缩肌肉纤维和肌原纤维中的肌球蛋白头的特定部位的光谱探针研究。附着在SH1(Cys 707，头部催化域)上的探针的电子顺磁共振(EPR)和时间分辨磷光各向异性(TPA)一致表明，收缩肌肉中的大多数肌球蛋白头是动态无序的，在微秒时间范围内经历大幅度旋转。其中一些无序的头部与肌动蛋白结合，特别是在ATPase周期的早期(弱结合、前)阶段。小的有序群体(10%-20%)严格定向，与严格一样，在收缩或存在被核苷酸类似物捕获的中间态时没有观察到其他明显的角度。这些结果与经典模型不一致，在经典模型中，整个头部在两个不同的方向之间经历了45度的转变。因此，有人提出肌球蛋白头部的催化结构域只有一个立体特异的(僵直的)肌动蛋白结合角度，并且在力产生过程中头部的内部结构发生变化，导致远端的轻链结合结构域旋转。为了测试这个模型，我们对附着在兔和扇贝的肌原纤维和纤维上的调控轻链(RLC)上的探针进行了EPR和TPA研究。RLC结果证实了动态(微秒)旋转紊乱在松弛和收缩中的优势，并表明两种肌肉中不同的钙调节机制产生了不同的旋转动力学。在兔肌原纤维中，RLC探针比SH1探针更动态地无序，特别是在僵硬和收缩方面，这表明当肌球蛋白头部与肌动蛋白相互作用时，轻链结合结构域经历相对于催化结构域的旋转运动。SH1结合的自旋标记对肌球蛋白的内部动力学很敏感，在收缩过程中分解三种不同的构象，时间分辨EPR表明这些转变与ATPase循环中的特定步骤相耦合。我们认为，在收缩过程中，力是通过无序到有序的转变产生的，在这种转变中，肌球蛋白头部首先以一种以大规模动态无序为特征的非立体特异性模式与肌动蛋白弱结合，然后经历至少两次构象转变，涉及头部内部的大规模结构(旋转)变化，最终形成一种高度有序的强结合状态。
We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the microsecond time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45 degree transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (microsecond) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction,indicating that the light-chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.