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: 7648218
• 负责人: SUSAN LOWEY
• 金额: $ 38.48万
• 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/7648218
• 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; Upper arm; Work; alanylproline; 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马达结构域的C-末端延伸的长α-螺旋区的旋转产生力和运动，并通过必需轻链（ELC）和调节轻链（RLC）稳定。轻链结合域或“杠杆臂”的作用是放大小的构象变化起源于核苷酸结合位点到更大的运动的杠杆arm. Despite最近的进展，在动力学和结构的方法，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然没有得到解决。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，以及不存在原子结构的肌动蛋白-肌球蛋白界面。在这里，我们提出了先进的技术，在电子低温显微镜（cryoEM）和图像分析，荧光显微镜和瞬态动力学提供进一步的见解机械化学耦合的机制。具体目标1将检查ELC的N-末端延伸与SH 3（src-同源3）结构域的结合。迄今为止，肌球蛋白中的SH 3样β-桶结构域的功能尚不清楚。我们将测试的假设，SH 3介导的ELC-1亚型，肌动蛋白和催化位点之间的通信途径，通过使用金标记的（cryoEM）和荧光标记（光谱）突变体表达ELC。ELC中的任何构象变化将通过停流动力学与ATP酶循环中的步骤相关。目的2将研究RLC的磷酸化如何导致平滑肌肌球蛋白从其抑制的去磷酸化状态激活。将通过将标记的半胱氨酸残基引入RLC和ELC中以便于通过荧光光谱法确定长度变化和相互作用位点来测试N-末端经历主要构象变化的假设。目的3将通过高分辨率cryoEM和新的计算方法，使用修饰有野生型和突变型心肌肌球蛋白亚型的肌动蛋白丝来表征肌动球蛋白界面。涉及轻链异常表达的病理生理条件，以及由肌球蛋白轻链和重链中的点突变引起的心肌病，将受益于对肌球蛋白中不同轻链和结构域如何相互作用以最大收缩效率进行工作的更深入理解。