MYOSIN DOMAIN INTERACTIONS DURING THE CONTRACTILE CYCLE

描述（由申请人提供）：Myosin被认为通过从运动结构域c端延伸的长a-螺旋区域的旋转产生力和运动，并由必需轻链（ELC）和调节轻链（RLC）稳定。轻链结合结构域或“杠杆臂”的作用是将源自核苷酸结合位点的小构象变化放大为杠杆臂的大运动。尽管最近在动力学和结构方法方面取得了进展，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。这部分是由于缺乏关于轻链的柔性n端区域的结构信息，以及不存在原子结构的肌动蛋白-肌凝蛋白界面。在此，我们提出了先进的电子冷冻显微镜和图像分析技术，荧光显微镜和瞬态动力学，以进一步了解机械化学耦合的机制。特异性目的1将研究ELC的n端延伸与SH3 （src-homology 3）结构域的结合。迄今为止，肌球蛋白中SH3-like （3-barrel）结构域的功能尚不清楚。我们将通过使用金标记（用于低温电镜）和荧光标记（用于光谱）表达的ELC突变体来验证SH3介导ELC-1异构体、肌动蛋白和催化位点之间的通讯途径的假设。通过停止流动动力学，ELC中的任何构象变化都与atp酶循环的步骤相关。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，去磷酸化状态激活。通过在RLC和ELC中引入标记的半胱氨酸残基来验证n端发生重大构象变化的假设，从而方便荧光光谱测定长度变化和相互作用位点。目的3将通过高分辨率冷冻电镜和新的计算方法来表征肌动蛋白界面，使用野生型和突变型心肌肌球蛋白异构体装饰的肌动蛋白丝。包括轻链异常表达的病理生理状况，以及由肌凝蛋白轻链和重链点突变引起的心肌病，将受益于对肌凝蛋白不同轻链和结构域如何相互作用以最大收缩效率工作的更深入理解。

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.