Protein Dynamics in Ca2+-Regulated Thin Filaments

超出所提供的空间。心肌收缩是由Ca2+依赖的蛋白相互作用的调节内的细丝。心肌研究的一个主要挑战是从细丝蛋白的结构和结构动力学的角度来理解这种调节。本研究计划继续研究肌肉收缩的机制，强调其调控背后的两个结构事件。第一个涉及Ca2*-心肌肌钙蛋白（cTn）介导的心肌肌钙蛋白I （cTnl）结构扰动，破坏cTnl-肌动蛋白复合物，第二个涉及细丝激活过程中心肌原肌球蛋白（cTm）的功能运动。我们假设，破坏cTnl-肌动蛋白复合物的cTnl结构变化与cTm的功能运动相耦合。这些结构和动态研究在肥厚性心肌病（HCM）的背景下具有特殊意义，其中cTm的单点突变以未知的机制导致心力衰竭。我们将通过测试某些HCM突变干扰薄细丝上cTm的功能构象转变的假设来探索其中的一些机制。该项目有四个具体目标：(1)建立cTnl上33个不同探针位点与肌动蛋白+/- ca2¿和肌凝蛋白固定位点之间的结构关系；(2)建立cTm c端10个不同探针位点相对于肌动蛋白+/- ca2¿和肌凝蛋白固定位点的结构关系。这些结构关系是通过高精度测量荧光共振能量转移（FRET）效率，取向因子/_，荧光极化（FP）在单个，Ca2+调控的灯丝，并通过拟合这些数据到薄灯丝的当前模型确定的；(3)表征Ca2+激活细丝时cTm内缓慢（ms-ms）的结构转变；(4)表征Ca2+激活细丝过程中特异性HCM突变对cTm结构动力学的影响。该研究的一个特别新颖的方面是，在松弛和Ca2+激活的细丝中，cTnl和cTm上的多个位点之间的结构关系将在人类心肌收缩的背景下建立。总的来说，这项工作的实施将促进我们对肌肉收缩分子基础的理解，并为肌肉收缩的机制提供新的见解。cTm中的特定突变导致心力衰竭。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Cardiac muscle contraction is regulated by a Ca2+-dependent modulation of protein interactions within the thin filament. A major challenge in cardiac muscle research is to understand this regulation in terms of the structures and structural dynamics of thin filament proteins. This research proposal continues investigations of the mechanism of muscle contraction emphasizing two structural events that underlie its regulation. The first involves the Ca2*-cardiac troponin (cTn) mediated structural perturbation in cardiac troponin I (cTnl) that disrupts the cTnl-actin complex, and the second involves functional movements of cardiac tropomyosin (cTm) during thin filament activation. We hypothesize that the structural changes in cTnl which disrupt the cTnl-actin complex are coupled to functional movements of cTm. These structural and dynamic studies take on special significance in the context of hypertrophic cardiomyopathy (HCM), where single point mutations in cTm lead to heart failure by unknown mechanisms. We will explore some of these mechanisms by testing the hypothesis that certain HCM mutations perturb functional conformational transitions of cTm on the thin filament. The project has four specific aims: (1), establish structural relationships between 33 different probe loci on cTnl with respect to a fixed locus on actin +/- Ca 2¿and myosin; (2), establish structural relationships between 10 different probe loci on the C-terminus of cTm with respect to a fixed locus on actin +/- Ca 2¿and myosin. These structural relationships are determined from high precision measurements of fluorescence resonance energy transfer (FRET) efficiency, orientation factor/_, fluorescence polarization (FP) on single, Ca2+-regulated filaments and by fitting these data into current models of the thin filament; (3), characterize slow (ms-ms) structural transitions within cTm during Ca2+-activation of thin filaments; (4), characterize the effects of specific HCM mutations on the structural dynamics of cTm during Ca2+-activation of thin filaments. A particularly novel aspect of the proposed research is that the structural relationships among multiple loci on cTnl and cTm in relaxed and Ca2+-activated thin filaments will be established in the context of human cardiac muscle contraction. Overall, implementation of the proposed work will advance our understanding of the molecular basis of muscle contraction and provide new insights into the mechanisms throu.clh which specific mutations in cTm lead to heart failure. PERFORMANCE SITE ========================================Section End===========================================