REGULATORY MECHANISMS OF CARDIAC MUSCLE

描述：（逐字摘自申请人摘要） 在肌肉收缩过程中产生的力量取决于 肌动蛋白和肌球蛋白之间的强相互作用。横纹肌中的这些相互作用 肌肉是由一个Ca（2+）调节的分子开关调节的， 肌钙蛋白（Tn）中Ca（2+）饱和水平和增强状态， 涉及通过力产生交叉桥的反馈控制。切换和 增强机制涉及三个Tn亚基之间的相互作用， Tn与原肌球蛋白（Tm）的相互作用。尽管对 在没有一个详细的法律程序的情况下， 蛋白质系统的三维结构，有可能获得 对分子相互作用的重要见解， 调节心肌肌丝的激活。这一切都是因为 根据最近在确定个体结构方面的进展， 蛋白质和阐明肌丝蛋白的排列。的 目前的研究解决了两个广泛的问题，这两个问题都与 开关和增强机制，并借鉴最近发表的 该实验室的荧光研究，持续的合作， 进入一个新的方向，和蛋白质的合作建模研究 组件.本课题有三个具体目标：（1）动力学比较 激活剂Ca（2+）与心脏和快速骨骼肌Tn结合的机制， 测定Ca（2+）诱导的调节细胞开放的动力学 （2）心脏Tn复合体的地形图绘制，以及 (3)Ca（2+）对纤维长度依赖性机理的探讨 张力激活和Ca（2+）诱导的构象变化 在等长张力发育过程中发生的调节蛋白。这些 研究将需要使用特定的突变蛋白。动力学研究 将通过停流荧光测定法进行。地形图将是 使用荧光共振能量转移（FRET）完成;两种标准 不同荧光团之间的异质转移和不同荧光团之间的同质转移 将使用相同的荧光团。目标3中的新方向将使用 皮肤肌肉纤维，并涉及同时测量张力和 由肌丝蛋白产生的荧光信号被交换成 细丝。这些研究的结果有望提高我们的 了解调节心肌肌丝变化的机制 对Ca（2+）的反应，并提供对钙离子的内在机制的见解。 心脏的控制由于许多心脏异常 涉及肌丝对Ca（2+）反应的变化和 调节蛋白，该项目的结果可能会提供深入了解 可能导致异常的分子事件

Description: (verbatim from the applicant's abstract) The magnitude of developed force during muscle contraction is dependent upon the number of strong interactions between actin and myosin. These interactions in striated muscle are modulated by a Ca(2+)-regulated molecular switch that determines the level of Ca(2+) saturation in troponin (Tn) and a potentiated state than involves a feedback control by force-generating crossbridges. The switching and potentiating mechanisms involve interactions among the three Tn subunits and interactions of Tn with tropomyosin (Tm). Although a description of the regulatory process cannot be formulated with detail in the absence of a three-dimensional structure of the protein system, it is possible to gain significant insights into the molecular interactions that are important in the regulation of activation of cardiac myofilaments. This has been made possible based on recent progress in the determination of the structure of individual proteins and elucidation of the arrangement of myofilament proteins. The present research addresses two broad issues that are related to both the switching and potentiating mechanisms and draws upon recently published fluorescence studies from this laboratory, a continued collaboration to allow entry into a new direction, and collaborative modeling studies of protein assemblies. This project has three specific aims: (1) comparison of the kinetic mechanisms by which activator Ca(2+) binds to cardiac and fast skeletal Tn and determination of the kinetics of Ca(2+)-induced opening of the regulatory domain in both isoforms, (2) topography mapping of the cardiac Tn complex, and (3) investigation of the mechanism of fiber length dependence of Ca(2+) activation of tension and Ca(2+)-induced conformational changes of the regulatory proteins that occur during isometric tension development. These studies will require the use of specific mutant proteins. The kinetic studies will be carried out by stopped-flow fluorometry. Topography mapping will be accomplished using fluorescence resonance energy transfer (FRET); both standard heterotransfer between different fluorophores and homotransfer between identical fluorophores will be used. The new direction in Aim 3 will use skinned muscle fibers and involve simultaneous measurement of tension and fluorescence signals arising from myofilament proteins which are exchanged into the filaments. Results from these studies are expected to enhance our understanding of the mechanisms that modulate changes in cardiac myofilament response to Ca(2+) and provide insights into intrinsic mechanisms of the control of the heart by Starling's law. Since many cardiac abnormalities appear to involve changes in myofilament response to Ca(2+) and altered structures of regulatory proteins, results from this project may provide insights into the molecular events that can contribute to abnormalities.