Smooth Muscle Myosin: Molecular Mechanics and Intramolecular Communication

描述(申请人提供)：每一个血管的管壁都有平滑肌细胞排列。正是它们的收缩功能对控制血压至关重要，当它们发生变化时，会导致高血压等疾病。在分子水平上，平滑肌收缩是肌球蛋白分子马达及其与肌动蛋白的循环相互作用的结果，这一过程是由肌球蛋白对ATP的水解提供动力的。肌球蛋白与横纹肌肌球蛋白的不同之处在于，它的肌球蛋白依赖于肌球蛋白的磷酸化调节和力量维持，消耗的能量(即ATP)很少。这项提议将研究平滑肌肌球蛋白的分子结构如何定义其机械性能。我们将通过使用杆状病毒表达系统将结构突变的力量与最先进的单分子生物物理技术(如激光陷阱)相结合，以评估肌球蛋白的双头结构如何有助于磷酸化依赖的调节。此外，还将设计突变肌球蛋白，以帮助确定每个平滑肌肌球蛋白的两个头部在产生最大力量和运动中的作用。所有肌肉对负荷的反应都是通过改变它们的缩短速度。因此，我们将确定肌球蛋白分子中感觉负荷的结构域，以及负荷如何调节肌球蛋白水解三磷酸腺苷的各个步骤。我们最初的重点将放在肌球蛋白转换和杠杆臂结构域上。我们还将利用在强直性(例如血管)和相性(例如肠)平滑肌肉中发现的自然存在的异构体，这两种肌肉具有显著不同的收缩特性，但其分子结构略有不同。不同之处在于肌球蛋白头部插入了7个氨基酸，以及两种基本的轻链异构体。这些肌球蛋白将通过对单个肌球蛋白分子施加负载，使用一种新的激光捕获力钳制分析来表征。拟议中的实验将为了解平滑肌维持血管张力的能力提供洞察力，而能量消耗很少。由于肌球蛋白分子马达存在于每一个平滑肌细胞中，并且与其他肌肉肌球蛋白有显著的相似之处，了解肌球蛋白的分子结构和功能不仅会影响我们如何治疗血管疾病，而且还会影响到心肌病。

DESCRIPTION (provided by applicant): Smooth muscle cells line the walls of every blood vessel. It is their contractile function that is critical to the control of blood pressure and when altered leads to diseases such as hypertension. At the molecular level, smooth muscle contraction is the result of the myosin molecular motor and its cyclic interaction with actin, a process powered by myosin's hydrolysis of ATP. Smooth muscle myosin is distinguished from the striated muscle myosins by its myosin phosphorylation-dependent regulation and force maintenance with little energy (i.e. ATP) expenditure. This proposal will investigate how smooth muscle myosin's molecular structure defines its mechanical performance. We will combine the power of structural mutagenesis through the use of the Baculovirus expression system with state-of-the-art single molecule biophysical techniques such as the laser trap to assess how myosin's double-headed structure contributes to phosphorylation-dependent regulation. In addition, mutant myosins will be designed that will help characterize the role of each of smooth muscle myosin's two heads in generating maximal force and motion. All muscles respond to load by varying their speed of shortening. Therefore, we will identify the structural domains within the smooth muscle myosin molecule that sense load and how load modulates the various steps of myosin's hydrolysis of ATP. Our initial focus will be on the myosin converter and lever arm domains. We will also take advantage of naturally occurring isoforms found in tonic (e.g. blood vessels) and phasic (e.g. intestine) smooth muscles, which have dramatically different contractile properties but with slight differences in their molecular structure. The differences are specifically a 7-amino acid insert in the myosin head and two essential light chain isoforms. These myosins will be characterized by applying load to single smooth muscle myosin molecules using a novel laser trap force clamp assay. The proposed experiments will provide insight to smooth muscle's ability to maintain vascular tone with little energy expenditure. Since the myosin molecular motor is found in every smooth muscle cell and shares significant similarities to other muscle myosins, understanding smooth muscle myosins molecular structure and function will impact not only how we may treat diseases of the vasculature but cardiomyopathies as well.