MOLECULAR MECHANICS OF MYOSIN FILAMENTS

这项研究的目的是量化个体效应 平滑肌和骨骼肌中的纤维结构和磷酸化 肌球蛋白，并检查它们在修改机械行为中的作用 单个肌球蛋白分子。 平滑肌肌球蛋白和骨骼肌肌球蛋白共同形成两种非常不同的 丝状结构 这种结构的功能后果 差异未知。 此外， 在关于肌球蛋白的“阶跃位移”的文献中， 估计范围为5 - 20 nm。所获得的值似乎 对应于在每一个中使用的肌球蛋白的结构状态， 实验室 总体而言，这些数据表明， 结构性组织对整体绩效至关重要， 肌球蛋白，肌丝内的肌球蛋白分子可以合作， 产生力和运动。 然而，分子间的协同性在 肌球蛋白丝的水平从未被系统地研究过， 因此这是本研究的第一个主要方向。 我们将 使用激光陷阱力传感器来测量和比较单个 平滑肌和骨骼肌产生的力和位移的阶跃 单体和丝状状态的肌球蛋白。 客观 是了解肌球蛋白丝的组成单体是否 合作，以及平滑肌和骨骼肌细丝是否表现出 在这方面不同。 肌球蛋白调节轻链的磷酸化改变了 平滑肌和骨骼肌中肌球蛋白丝的结构。 正因为如此，我们有义务检查磷酸化和 纤维结构，以了解任何一个。 结构变化 在磷酸化后发生的，似乎是一种延伸， 肌球蛋白离开肌球蛋白丝并朝向肌动蛋白。 这 构象变化可能是建立良好的调节， 收缩平滑肌磷酸化，以及 增强骨骼肌的力量产生。 但 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，第二个主要推力 这项研究的一个目的是研究磷酸化 这些肌球蛋白在其运动过程中产生的力和位移 单体和丝状状态。 这些数据对我们的 了解肌球蛋白如何在分子水平上发挥作用 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.