Thomas D D, Ramachandran S, Roopnarine O, Hayden D W, Ostap E M
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biophys J. 1995 Apr;68(4 Suppl):135S-141S.
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.
我们提出了一种肌肉中力产生的分子机制，主要基于对收缩肌纤维和肌原纤维中肌球蛋白头部的位点特异性光谱探针研究。附着于SH1（头部催化结构域中的Cys 707）的探针的电子顺磁共振（EPR）和时间分辨磷光各向异性（TPA）一直表明，收缩肌肉中的大多数肌球蛋白头部是动态无序的，在微秒时间范围内进行大幅度旋转。其中一些无序头部与肌动蛋白结合，特别是在ATP酶循环的早期（弱结合、预力）阶段。小部分有序群体（10 - 20%）严格地定向，就像在强直收缩状态一样，在收缩过程中或存在核苷酸类似物捕获的中间状态时未观察到其他明显角度。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方向之间经历45度转变。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性（强直收缩样）肌动蛋白结合角，并且在力产生过程中头部的内部结构发生变化，导致远端轻链结合结构域旋转。为了验证该模型，我们对兔和扇贝肌原纤维及纤维中附着于调节轻链（RLC）的探针进行了EPR和TPA研究。RLC结果证实了在松弛和收缩过程中动态（微秒）旋转无序的主导地位，并表明两种肌肉中钙调节的不同机制产生不同旋转动力学。在兔肌原纤维中，RLC探针比SH1探针更具动态无序性，特别是在强直收缩和收缩状态下，这表明当肌球蛋白头部与肌动蛋白相互作用时，轻链结合结构域相对于催化结构域进行旋转运动。一个附着于SH1的自旋标记物，对肌球蛋白的内部动力学敏感，在收缩过程中分辨出三种不同构象，时间分辨EPR表明这些转变与ATP酶循环中的特定步骤相关联。我们提出，在收缩过程中力是由无序到有序的转变产生的，其中肌球蛋白头部首先以非立体特异性模式弱附着于肌动蛋白，其特征是大规模动态无序，然后经历至少两次构象转变，涉及头部内大规模结构（旋转）变化，最终形成承受力的高度有序的强结合状态。
