Protein Dynamics in Ca2+-Regulated Thin Filaments

超出提供的空间。心肌收缩受钙依赖的细丝内蛋白质相互作用的调节。心肌研究中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。这项研究计划继续研究肌肉收缩的机制，强调了两个结构性事件，它们是调节肌肉收缩的基础。第一个涉及钙离子-心肌肌钙蛋白(CTn)介导的心肌肌钙蛋白I(CTnl)结构扰动，破坏cTn1-肌动蛋白复合体；第二个涉及心肌原肌球蛋白(CTM)在细丝激活过程中的功能性运动。我们假设cTn1中破坏cTn1-肌动蛋白复合体的结构变化与CTM的功能运动相耦合。这些结构和动态研究在肥厚型心肌病(HCM)的背景下具有特殊的意义，其中CTM的单点突变导致心力衰竭的机制未知。我们将通过测试某些HCM突变扰乱细丝上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cTn1上33个不同探针座位与肌动蛋白/-钙离子和肌球蛋白之间的结构关系；(2)建立中药C端10个不同探针座位与肌动蛋白/-钙离子和肌球蛋白固定位点之间的结构关系。这些结构关系是通过高精度测量单个钙调控的细丝的荧光共振能量转移(FRET)效率、取向因子/_、荧光偏振(FP)并将这些数据拟合到当前的细丝模型来确定的；(3)表征了细丝在钙激活过程中CTM内缓慢的(ms-ms)结构转变；(4)表征了特定的HCM突变对细丝在钙激活过程中CTM结构动力学的影响。这项研究的一个特别新颖的方面是，在松弛和钙激活的细丝中，cTn1和CTM上的多个基因座之间的结构关系将在人类心肌收缩的背景下建立。总体而言，这项拟议工作的实施将促进我们对肌肉收缩的分子基础的理解，并为通过CTM特定突变导致心力衰竭的机制提供新的见解。表演网站========================================Section End===========================================

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===========================================