Smooth Muscle Myosin: Molecular Mechanics and Intramolecular Communication

平滑肌肌球蛋白：分子力学和分子内通讯

• 批准号: 7275965
• 负责人: David M Warshaw
• 金额: $ 36.9万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7275965
• 关键词: ATP Hydrolysis; Actins; Active Sites; Actomyosin Adenosinetriphosphatase; Affect; Affinity; Amino Acids; Arts; Baculovirus Expression System; Binding; Biological Assay; Blood Vessels; Cardiomyopathies; Cell Line; Communication; Dependence; Disease; Elements; Energy Metabolism; Expenditure; Head; Hypertension; Intestines; Kinetics; Lasers; Light; Maintenance; Measures; Mechanics; Molecular; Molecular Motors; Molecular Structure; Motion; Muscle; Muscle Contraction; Mutagenesis; Mutate; Myosin ATPase; Neck; Pathway interactions; Performance; Phosphorylation; Point Mutation; Process; Property; Protein Isoforms; Regulation; Research Personnel; Role; Skeletal Muscle Myosins; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Speed; Striated Muscles; Structure; Techniques; Upper arm; Work; blood pressure regulation; design; insight; molecular mechanics; mutant; novel; programs; research study; single molecule

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.

描述（由申请人提供）：平滑肌细胞排列在每条血管的壁上。它们的收缩功能对于控制血压至关重要，一旦改变就会导致高血压等疾病。在分子水平上，平滑肌收缩是肌球蛋白分子马达及其与肌动蛋白循环相互作用的结果，这一过程由肌球蛋白水解 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力维持，且能量（即 ATP）消耗很少。该提案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诱变的力量与最先进的单分子生物物理技术（例如激光陷阱）结合起来，以评估肌球蛋白的双头结构如何促进磷酸化依赖性调节。此外，突变肌球蛋白的设计将有助于表征平滑肌肌球蛋白的两个头在产生最大力和运动方面的作用。所有肌肉都通过改变缩短速度来响应负荷。因此，我们将确定平滑肌肌球蛋白分子内感知负荷的结构域以及负荷如何调节肌球蛋白 ATP 水解的各个步骤。我们最初的重点将是肌球蛋白转换器和杠杆臂领域。我们还将利用在强直（例如血管）和阶段性（例如肠道）平滑肌中发现的天然同工型，它们具有显着不同的收缩特性，但分子结构略有不同。具体差异在于肌球蛋白头部的 7 个氨基酸插入和两个必需的轻链亚型。这些肌球蛋白将通过使用新型激光陷阱力钳测定对单个平滑肌肌球蛋白分子施加负载来表征。拟议的实验将深入了解平滑肌以很少的能量消耗维持血管张力的能力。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着相似性，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脉管系统疾病，还会影响心肌病。