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根血管的壁上。正是它们的收缩功能对控制血压至关重要，一旦改变就会导致高血压等疾病。在分子水平上，平滑肌收缩是肌凝蛋白分子马达及其与肌动蛋白循环相互作用的结果，这一过程由肌凝蛋白水解ATP提供动力。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维持力，而能量（即ATP）消耗很少。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。我们将结合结构诱变的力量，通过使用杆状病毒表达系统和最先进的单分子生物物理技术，如激光陷阱，来评估肌球蛋白的双头结构如何有助于磷酸化依赖性调节。此外，将设计突变型肌球蛋白，这将有助于描述平滑肌肌球蛋白的两个头在产生最大力量和运动中的作用。所有的肌肉都以不同的收缩速度对负荷作出反应。因此，我们将确定平滑肌肌球蛋白分子中感知负荷的结构域，以及负荷如何调节肌球蛋白水解ATP的各个步骤。我们最初的重点将放在肌凝蛋白转化器和杠杆臂结构域。我们还将利用在强直肌（如血管）和相位性平滑肌（如肠）中发现的自然存在的同种异构体，它们具有显著不同的收缩特性，但分子结构略有不同。不同之处在于肌凝蛋白头部的7个氨基酸插入和两个基本的轻链异构体。这些肌球蛋白将通过使用新型激光诱捕力钳法对单个平滑肌肌球蛋白分子施加负载来表征。提出的实验将提供洞察平滑肌维持血管张力的能力，以很少的能量消耗。由于肌球蛋白分子运动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将影响我们如何治疗血管疾病，也将影响我们如何治疗心肌病。

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.