Smooth Muscle Myosin: Molecular Mechanics and Intramolecular Communication

描述（由申请人提供）：平滑肌细胞排列在每个血管的壁上。正是它们的收缩功能对控制血压至关重要，当改变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，这是由肌球蛋白水解ATP提供动力的过程。平滑肌肌球蛋白与横纹肌肌球蛋白的区别在于其肌球蛋白磷酸化依赖性调节和力维持，几乎没有能量（即ATP）消耗。本研究将探讨平滑肌肌球蛋白的分子结构如何决定其机械性能。我们将结合联合收割机的力量，通过使用杆状病毒表达系统与国家的最先进的单分子生物物理技术，如激光陷阱，以评估肌球蛋白的双头结构如何有助于磷酸化依赖性调节的结构诱变。此外，突变肌球蛋白将被设计，这将有助于表征平滑肌肌球蛋白的两个头部中的每一个在产生最大的力和运动中的作用。所有肌肉对负荷的反应都是通过改变缩短的速度来实现的。因此，我们将确定平滑肌肌球蛋白分子内的结构域，感觉负荷和负荷如何调节肌球蛋白水解ATP的各个步骤。我们最初的重点将是肌球蛋白转换器和杠杆臂结构域。我们还将利用在强直性（例如血管）和阶段性（例如肠）平滑肌中发现的天然存在的同种型，其具有显著不同的收缩特性，但其分子结构略有差异。差异具体是肌球蛋白头中的7个氨基酸插入物和两种必需的轻链同种型。这些肌球蛋白将通过使用新型激光陷阱力钳测定法对单个平滑肌肌球蛋白分子施加负载来表征。拟议的实验将提供洞察平滑肌的能力，以维持血管张力与很少的能量消耗。由于肌球蛋白分子马达存在于每个平滑肌细胞中，并且与其他肌肉肌球蛋白具有显著的相似性，因此了解平滑肌肌球蛋白分子结构和功能不仅会影响我们如何治疗血管疾病，还会影响心肌病。

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.