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）：平滑肌细胞在每个血管的壁上排列。他们的收缩功能对控制血压至关重要，而当改变会导致诸如高血压之类的疾病。在分子水平上，平滑肌收缩是肌球蛋白分子运动的结果及其与肌动蛋白的循环相互作用，这是由肌球蛋白对ATP的水解提供动力的过程。平滑肌肌球蛋白通过肌球蛋白磷酸化依赖性调节和力量维持力（即ATP）支出而与肌肉肌球蛋白的肌球蛋白磷酸化依赖性调节和力量维持区分开来。该建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与最先进的单分子生物物理技术（例如激光陷阱）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，从而结合结构诱变的力量。此外，将设计突变体肌球蛋白，这将有助于表征平滑肌肌球蛋白两个头部在产生最大力和运动中的作用。所有肌肉都通过改变其缩短速度来应对负载。因此，我们将确定平滑肌肌球蛋白分子中的结构结构域，这些结构域感应负荷以及负载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球蛋白转换器和杠杆臂域上。我们还将利用在补品（例如血管）和阶段（例如肠道）平滑肌中发现的自然存在的同工型，它们具有截然不同的收缩特性，但其分子结构却有很小的差异。这些差异是在肌球蛋白头部和两个必需轻链同工型中特别的7-氨基酸插入仪。这些肌球蛋白的特征是使用新型激光陷阱力夹测定法对单平滑肌肌球蛋白分子施加载荷。提出的实验将为平滑肌的能力提供洞察力，使肌肉能够保持血管张力的能力，而能量消耗很少。由于在每个平滑肌细胞中都发现了肌球蛋白分子运动，并且与其他肌肉肌球蛋白具有显着相似之处，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的疾病，还会影响心肌病的疾病。