MYOSIN DOMAIN INTERACTIONS DURING THE CONTRACTILE CYCLE

收缩周期期间肌球蛋白域的相互作用

• 批准号: 7874572
• 负责人: SUSAN LOWEY
• 金额: $ 39.24万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7874572
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Active Sites; Actomyosin; Affect; Binding; Binding Sites; Biological Models; C-terminal; Cardiac; Cardiac Myosins; Cardiomyopathies; Catalytic Domain; Communication; Computing Methodologies; Coupling; Cryoelectron Microscopy; Cysteine; Engineering; Fluorescence Microscopy; Fluorescence Spectroscopy; Gold; Head; Image Analysis; Imaging Techniques; Individual; Kinetics; Label; Lead; Length; Light; Mediating; Microfilaments; Molecular Motors; Monitor; Motor; Movement; Muscle; Muscle Rigidity; Mutation; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; N-terminal; Nucleic Acid Regulatory Sequences; Nucleotides; Pathway interactions; Phosphorylation; Point Mutation; Positioning Attribute; Power stroke; Protein Biochemistry; Protein Isoforms; Research Personnel; Resolution; Role; Rotation; Site; Smooth Muscle; Smooth Muscle Myosins; Spectrum Analysis; Striated Muscles; Structure; Techniques; Testing; Transgenic Mice; Work; alanylproline; arm; base; flexibility; insight; mutant; programs

DESCRIPTION (provided by applicant): Myosin is believed to generate force and movement by the rotation of a long a-helical region that extends from the C-terminus of the motor domain, and is stabilized by the essential light chain (ELC) and the regulatory light chain (RLC). The role of the light chain-binding domain or "lever arm" is to amplify small conformational changes originating at the nucleotide binding site into larger movements of the lever arm. Despite recent advances in kinetic and structural approaches, many aspects of the communication pathway between ATP hydrolysis, actin-binding, and the lever arm remain unresolved. This is due, in part, from an absence of structural information regarding the flexible N-terminal regions of the light chains, and the actin- myosin interface for which no atomic structure exists. Here we propose advanced techniques in electron cryomicroscopy (cryoEM) and image analysis, fluorescence microscopy and transient kinetics to provide further insights into the mechanism of mechanochemical coupling. Specific Aim 1 will examine the binding of the N-terminal extension of ELC to the SH3 (src-homology 3) domain. To date, the function of the SH3-like (3-barrel domain in myosin is unknown. We will test the hypothesis that SH3 mediates the communication pathway between the ELC-1 isoform, actin, and the catalytic site, by using gold-labeled- (for cryoEM) and fluorescent-labeled (for spectroscopy) mutants of expressed ELC. Any conformational changes in ELC will be correlated with steps in the ATPase cycle by stopped-flow kinetics. Aim 2 will examine how phosphorylation of the RLC leads to activation of smooth muscle myosin from its inhibited, dephosphorylated state. The hypothesis that the N-terminus undergoes a major conformational change will be tested by introducing labeled cysteine residues into RLC and ELC to facilitate determination of length changes and sites of interaction by fluorescence spectroscopy. Aim 3 will characterize the actomyosin interface by high resolution cryoEM, and new computational methodologies, using actin filaments decorated with wild type and mutant cardiac myosin isoforms. Pathophysiological conditions involving abnormal expression of the light chains, as well as cardiomyopathies resulting from point mutations in the light and heavy chains of myosin, will benefit from a deeper understanding of how the different light chains and domains in myosin interact to perform work with maximum contractile efficiency.

描述（由申请人提供）：据信肌球蛋白通过从运动结构域的C末端延伸的长A螺旋区域的旋转来产生力和运动，并通过必需的轻链（ELC）和调节光链（RLC）稳定。轻链结合结构域或“杠杆臂”的作用是扩大源自核苷酸结合位点的小构象变化到杆臂的较大运动中。尽管动力学和结构方法最近取得了进步，但ATP水解，肌动蛋白结合和杠杆臂之间的通信途径的许多方面仍未解决。这部分是由于缺乏有关光链的柔性N末端区域以及不存在原子结构的肌动蛋白肌球蛋白界面的原因。在这里，我们提出了电子冷冻显微镜（冷冻）和图像分析，荧光显微镜和瞬时动力学的高级技术，以进一步了解机械化学偶联机理。具体目标1将检查ELC的N末端扩展与SH3（SRC-同源3）结构域的结合。 To date, the function of the SH3-like (3-barrel domain in myosin is unknown. We will test the hypothesis that SH3 mediates the communication pathway between the ELC-1 isoform, actin, and the catalytic site, by using gold-labeled- (for cryoEM) and fluorescent-labeled (for spectroscopy) mutants of expressed ELC. Any conformational changes in ELC will be correlated with steps in the ATPase循环通过停止流动动力学2。荧光光谱法将使用用野生型和突变的心脏肌球蛋白同工型装饰的肌动蛋白丝，通过高分辨率冷冻素和新的计算方法来表征Actomyosin的界面。涉及光链异常表达的病理生理状况以及肌球蛋白的光和重链中的点突变引起的心肌病，将受益于对肌球蛋白在肌球蛋白相互作用中如何相互作用以与最大收缩效率一起工作的更深入了解。