Protein Dynamics in Ca2+-Regulated Thin Filaments

Ca2 调节细丝中的蛋白质动力学

• 批准号: 6822621
• 负责人: GERARD MARRIOTT
• 金额: $ 28.86万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6822621
• 关键词: actins; calcium ion; fluorescence polarization; fluorescence resonance energy transfer; heart contraction; hypertrophic myocardiopathy; laboratory rabbit; microfilaments; muscle contraction; myocardium; myosins; peptide library; protein purification; protein structure function; tropomyosin; troponin

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

超出所提供的空间。心肌收缩是由Ca 2+依赖性调节蛋白质相互作用的细丝。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方面理解这种调节。这项研究计划继续调查肌肉收缩的机制，强调两个结构性事件，其监管的基础。第一个涉及Ca 2 *-心肌肌钙蛋白（cTnI）介导的结构扰动，破坏cTnI-肌动蛋白复合物，第二个涉及心肌原肌球蛋白（cTm）在细丝激活过程中的功能运动。我们假设破坏cTnl-肌动蛋白复合物的cTnl结构变化与cTm的功能性运动相关联。这些结构和动力学研究在肥厚型心肌病（HCM）的背景下具有特殊意义，其中cTm中的单点突变通过未知机制导致心力衰竭。我们将探讨这些机制，通过测试的假设，某些HCM突变扰动的cTm的功能构象转变的细丝。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+/- Ca 2 <$和肌球蛋白上固定位点之间的结构关系;（2）建立cTm C端上10个不同探针位点与肌动蛋白+/- Ca 2 <$和肌球蛋白上固定位点之间的结构关系。这些结构关系是通过对单个Ca 2+调节的细丝上的荧光共振能量转移（FRET）效率、取向因子f_、荧光偏振（FP）的高精度测量以及通过将这些数据拟合到细丝的当前模型中来确定的;（3）表征细丝的Ca 2+激活期间cTm内的缓慢（ms-ms）结构转变;（4）表征在细丝的Ca 2+激活期间特定HCM突变对cTm的结构动力学的影响。所提出的研究的一个特别新颖的方面是，将在人心肌收缩的背景下建立放松和Ca 2+激活的细丝中cTnl和cTm上的多个位点之间的结构关系。总的来说，所提出的工作的实施将推进我们对肌肉收缩的分子基础的理解，并提供对cTm中特定突变导致心力衰竭的机制的新见解。性能现场=