MOLECULAR MECHANICS OF MYOSIN FILAMENTS

这项研究的目标是量化个体效应。 平滑肌和骨骼肌的细丝结构和磷酸化 肌球蛋白，并研究它们在改变机械行为中的作用 单个肌球蛋白分子。 平滑肌球蛋白和骨骼肌球蛋白聚合形成两个非常不同的 细丝结构。这一结构的功能后果 差异是未知的。此外，还存在巨大的分歧。 在有关肌球蛋白“阶跃位移”的文献中， 估计从5到20纳米不等。所获得的值似乎是 与肌球蛋白的结构状态相对应 实验室。总体而言，这些数据表明， 结构性组织对企业的整体绩效至关重要 肌球蛋白，而细丝中的肌球蛋白分子可以协同作用 产生力量和运动。然而，分子间的协作性在 肌球蛋白细丝的水平从未被系统地研究过， 因此，这是拟议研究的第一个主要推动力。我们会 使用激光捕捉力传感器测量和比较单个 由平滑和骨骼肌产生的力的步数和位移 肌球蛋白既有单体状态，也有丝状状态。目标是 就是了解肌球蛋白细丝的组成单体是否起作用 协同作用，以及平滑和骨骼肌丝是否表现出 在这方面有所不同。 肌球蛋白调节轻链的磷酸化改变 肌球蛋白细丝在平滑肌肉和骨骼肌中的结构。 正因为如此，我们有义务检查磷酸化和 为了更好的了解花丝的结构。结构性变化 在磷酸化时发生的，似乎是 肌球蛋白从肌球蛋白细丝向肌动蛋白进发。这 构象变化可能是既定的规则的基础 通过磷酸化引起的平滑肌收缩，以及 增强骨骼肌力的产生。然而， 磷酸化的机械后果还有待研究。 单个肌球蛋白头部的水平。因此，第二个主要推动力 这项拟议的研究的目的是检查磷酸化的影响 关于这些肌球蛋白在其体内产生的力和位移 单体状态和丝状状态。这些数据对我们的 肌球蛋白在整体分子水平上的功能 细胞和组织。

The goal of the proposed research is to quantify the individual effects of filament structure and phosphorylation in smooth and skeletal muscle myosins, and to examine their roles in modifying the mechanical behavior of single myosin molecules. Smooth and skeletal muscle myosins polymerize to form two very distinct filament structures. The functional consequence of this structural difference is unknown. Furthermore, there is tremendous disagreement in the literature as to the "step displacement" of myosin, with estimates ranging from 5 - 20 nm. The value obtained appears to correspond to the structural state of the myosin being used in each laboratory. Taken as a whole, these data suggest that the level of structural organization is critical to the overall performance of myosin, and that myosin molecules within a filament may cooperate to generate force and motion. However, intermolecular cooperativity at the level of myosin filaments has never been systematically investigated, and is thus the first major thrust of the proposed research. We will use a laser trap force transducer to measure and compare the single steps in force and displacement generated by smooth and skeletal muscle myosins both in their monomeric and filamentous states. The objective is to understand if the constituent monomers of myosin filaments act cooperatively, and whether smooth and skeletal muscle filaments behave differently in this respect. Phosphorylation of the regulatory light chain of myosin alters the structures of myosin filaments in both smooth and skeletal muscles. Because of this, we are obligated to examine both phosphorylation and filament structure in order to understand either. The structural change that occurs upon phosphorylation appears to be an extension of the myosin heads away from the myosin filament and toward actin. This conformational change may underlie the well-established regulation of contraction in smooth muscle by phosphorylation, as well as the enhancement of force generation in skeletal muscle. However, the mechanical consequences of phosphorylation have yet to be examined at the level of the individual myosin heads. Thus, the second major thrust of the proposed research is to examine the effects of phosphorylation on the forces and displacements generated by these myosins in their monomeric and filamentous states. These data are critical to our understanding of how myosin functions at the molecular level in whole cells and tissues.