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: 7455162
• 负责人: SUSAN LOWEY
• 金额: $ 37.36万
• 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/7455162
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Active Sites; Actomyosin; Affect; Altretamine; Binding; Binding Sites; Biological Models; C-terminal; Cardiac; Cardiac Myosins; Cardiomyopathies; Catalytic Domain; Class; Communication; Computing Methodologies; Condition; Coupling; Cryoelectron Microscopy; Cysteine; Depth; 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; Upper arm; Work; alanylproline; base; 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(申请人提供)：肌球蛋白被认为通过旋转一个从运动域的C末端延伸的长的a-螺旋区域来产生力量和运动，并由基本轻链(ELC)和调节轻链(RLC)稳定。轻链结合域或“杠杆臂”的作用是将起源于核苷酸结合部位的小构象变化放大为杠杆臂的较大运动。尽管最近在动力学和结构方法上取得了进展，但ATP水解、肌动蛋白结合和杠杆臂之间的通讯途径的许多方面仍然没有解决。这部分是由于缺乏关于轻链的柔性N-末端区域的结构信息，以及不存在其原子结构的肌动蛋白-肌球蛋白界面。在这里，我们介绍了电子冷冻显微镜(CryoEM)和图像分析、荧光显微镜和瞬时动力学方面的先进技术，以进一步深入了解机械力化学耦合的机制。特定目的1将检查ELC的N-末端延伸与SH3(src-Homology 3)结构域的结合。到目前为止，肌球蛋白中SH3-like(3-Barrel)结构域的功能尚不清楚。我们将通过使用表达的ELC的金标记突变体(用于低温EM)和荧光标记突变体(用于光谱学)来验证SH3介导ELC-1异构体、肌动蛋白和催化位点之间的通信途径的假设。ELC中的任何构象变化都将通过停流动力学与ATPase循环中的步骤相关联。目的2将研究RLC的磷酸化如何导致平滑肌肌球蛋白从被抑制的去磷酸化状态激活。N末端发生主要构象变化的假设将通过将标记的半胱氨酸残基引入RLC和ELC来检验，以便于用荧光光谱确定长度变化和相互作用位置。目的3将用高分辨率的低温电子显微镜和新的计算方法来表征肌动球蛋白的界面，使用野生型和突变型心肌肌球蛋白亚型装饰的肌动蛋白细丝。涉及轻链异常表达的病理生理条件，以及肌球蛋白轻链和重链的点突变导致的心肌病，将有助于更深入地了解肌球蛋白中不同的轻链和结构域如何相互作用，以最大限度地收缩效率。