REGULATORY MECHANISMS OF CARDIAC MUSCLE

描述：(逐字摘自申请者的摘要) 肌肉收缩时产生的力量取决于肌肉收缩的数量 肌动蛋白和肌球蛋白之间的强相互作用。这些相互作用是横纹的 肌肉受钙(2+)调节的分子开关调节，决定了 肌钙蛋白(Tn)中Ca(2+)饱和度的水平及其增强状态 包括通过产生力的交叉桥进行反馈控制。切换和 增强机制涉及三个TN亚基之间的相互作用和 TN与原肌球蛋白(TM)的相互作用尽管对 监管流程在缺乏 蛋白质体系的三维结构，就有可能获得 对分子相互作用的重要见解 心肌肌丝激活的调控。这已经成为可能 根据最近在确定个体结构方面的进展 蛋白质和肌丝蛋白质排列的阐明。这个 本研究涉及两个广泛的问题，这两个问题都与 开关和增强机制并借鉴了最近发表的 来自该实验室的荧光研究，继续合作，以允许 进入新的方向，蛋白质的协同建模研究 装配。本项目有三个具体目标：(1)比较动力学 激活剂Ca(2+)与心肌和快速骨骼Tn结合的机制 钙离子诱导的调节性钙通道开放动力学的测定 两种异构体中的结构域，(2)心脏TN复合体的地形图，以及 (3)研究了Ca(2+)对纤维长度的依赖机理 张力的激活和Ca(2+)诱导的构象变化 在等长张力发育过程中出现的调节蛋白。这些 研究将需要使用特定的突变蛋白。动力学研究 将用停流荧光法进行测定。地形图绘制将是 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同质转移 将使用相同的荧光团。《目标3》中的新方向将使用 去皮的肌肉纤维，包括同时测量张力和 来自肌丝蛋白的荧光信号被交换到 细丝。这些研究的结果可望加强我们的 对心肌肌丝变化调控机制的认识 对Ca(2+)的响应，并提供对内源性机制的见解 斯塔林定律对心脏的控制。由于出现了许多心脏异常 涉及肌丝对Ca(2+)反应的变化和心肌细胞结构的改变 调控蛋白，这个项目的结果可能会为我们提供对 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.