Diversity Supplement to Structural Dynamics of Cardiac Myosin-Binding Protein C Regulation

心肌肌球蛋白结合蛋白 C 调节结构动力学的多样性补充

• 批准号: 10412720
• 负责人: Brett A Colson
• 金额: $ 3.15万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412720
• 关键词: Accounting; Actins; Address; Affect; Anisotropy; Binding; Binding Proteins; Biological Assay; Biological Models; Biophysical Process; Biophysics; Cardiac; Cardiac Myosins; Cardiomyopathies; Cardiovascular Diseases; Complex; Computer Simulation; Detection; Development; Diastole; Disease; Drug Screening; Equilibrium; FDA approved; Familial Hypertrophic Cardiomyopathy; Filament; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Foundations; Goals; Health; Heart; Heart Diseases; Heart failure; Human; Hypertrophic Cardiomyopathy; In Vitro; Individual; Kinetics; Knowledge; Label; Lead; Learning; Location; Measures; Medical; Methods; Microfilaments; Molecular; Molecular Conformation; Molecular Structure; Muscle; Muscle Cells; Muscle Proteins; Muscle function; Mutation; Myocardial; Myocardium; Myosin ATPase; N-terminal; Parents; Pathogenicity; Pathologic; Pathology; Performance; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phosphorylation; Physiological; Physiology; Play; Positioning Attribute; Property; Proteins; Publishing; Recombinants; Regulation; Reporter; Research; Research Project Grants; Role; Sarcomeres; Site; Spectrum Analysis; Stress; Structure; Systole; Technology; Testing; Therapeutic; Thick; Thick Filament; Thin Filament; Thinness; Time; Training; Tropomyosin; Troponin; Work; base; biophysical model; cardiac muscle disease; career development; design; effective therapy; heart function; improved; innovation; insight; mutant; myosin-binding protein C; nanometer; next generation; novel; novel therapeutics; parent grant; phosphorescence; prevent; protein complex; protein structure; reconstitution; screening; skills; spectroscopic data; spectroscopic survey; symptom management; therapeutic target; time use; tool

Title of project: DIVERSITY SUPPLEMENT TO STRUCTURAL DYNAMICS OF CARDIAC MYOSIN-BINDING PROTEIN C REGULATION. Myosin-binding protein C (MyBP-C) plays a major role in the modulation of cardiac function by its phosphorylation and causes deficits in contractile function due to MyBP-C mutations in hypertrophic cardiomyopathy (HCM) and reduced phosphorylation in heart failure. Our goal is to understand the molecular biophysics of muscle, with particular emphasis on the heart, and to train the next generation of muscle biophysicists, inclusive of diverse trainees. The parent research project and diversity supplement ask fundamental questions about the role of protein interactions and structural dynamics that regulate function in cardiac muscle. To gain insight into the correlation of structure-function involved in MyBP-C mechanisms in physiological and pathological settings, we will probe the actin-myosin-MyBP-C complex of these proteins in solution with varied binding, phosphorylation, and HCM mutations. Our core technology is site-directed spectroscopy, applied to purified MyBP-C and actin/myosin filaments. We will apply innovative complementary methods in site-directed labeling and spectroscopy to correlate protein binding, structural dynamics and function. We will test the central hypothesis that phosphorylation and HCM mutations of N-terminal MyBP-C alter functionally significant structural properties of MyBP-C alone and as it interacts with actin and myosin. Related to parent grant Aim 1, the first period of the diversity supplement focuses on using spectroscopic approaches to accurately measure the structural dynamics within M-domain of purified MyBP-C, where phosphorylation occurs, primarily by measuring nanometer distances and molecular disorder. Major emphasis is placed on detection of conformational changes (structure) within MyBP-C’s M-domain due to phosphorylation, HCM mutation, and actin or myosin binding (function). By including the location of probes in M-domain, the Candidate will measure structural changes predicted from our computational simulations. Fluorescently-labeled MyBP-C will be prepared to acquire fluorescence lifetime using time-resolved methods. In the second period, the Candidate will learn new skills in spectroscopic data fitting analysis to determine probe-to-probe distances and disorder in N-terminal MyBP-C. The third period will contribute to both Aim 1 and Aim 2 by providing molecular details of the structural dynamics of the actin-MyBP-C complex with added troponin, tropomyosin, and myosin to mimic physiological conditions of the cardiac thin filament in muscle. The Candidate will systematically build in model system complexity, from actin-bound to thin filament-bound MyBP-C, with low (diastole) or high (systole) activator Ca2+, and upon binding of cardiac myosin, providing key insights at the myofilament level to be applied for understanding fundamental mechanisms in the muscle cell. Spectroscopic study of MyBP-C regulation of thin filaments will determine protein interactions and structural dynamics. This project is grounded in fundamental biophysics mechanisms, but MyBP-C has emerged as a therapeutic target for cardiac disease. Thus, of our work lays a foundation for development of screens for drug therapies using our unique spectroscopic approaches.

项目标题：心脏肌球蛋白结合结构动力学的多样性补充 蛋白C调节。 肌球蛋白结合蛋白C（MYBP-C）在心脏功能调节中起主要作用 磷酸化并导致由于肥厚型MYBP-C突变引起的收缩功能的定义 心肌病（HCM）和心力衰竭的磷酸化降低。我们的目标是了解分子 肌肉的生物物理学，特别强调心脏，并训练下一代肌肉 生物物理学家，包括多样性学员。家长研究项目和多样性补充要求 关于调节功能的蛋白质相互作用和结构动力学作用的基本问题 心肌。洞悉MYBP-C机制中涉及的结构功能的相关性 物理和病理环境，我们将探测这些蛋白质的肌动蛋白 - 肌球蛋白 - 米型-C-C复合物 具有不同结合，磷酸化和HCM突变的溶液。我们的核心技术是定向的 光谱法，用于纯化的MYBP-C和肌动蛋白/肌球蛋白丝。我们将应用创新的完件 位置定向的标记和光谱法中的方法，可将蛋白质结合，结构动力学和功能相关联。 我们将测试中心假设，即N末端MYBP-C Alter的磷酸化和HCM突变 单独使用MYBP-C并与肌动蛋白和肌球蛋白相互作用，在功能上具有重要的结构特性。有关的 对于父母授予目标1，多样性补充的第一阶段侧重于使用光谱方法 准确测量纯化的MYBP-C的M域内的结构动力学，其中发生磷酸化， 首先，通过测量纳米距离和分子障碍。主要重点是检测 由于磷酸化，HCM突变和肌动蛋白 或肌球蛋白结合（功能）。通过在M域中包括问题的位置，候选人将测量 从我们的计算模拟预测的结构变化。将准备荧光标记的MYBP-C 使用时间分辨方法获取荧光寿命。在第二阶段，候选人将学习新的 光谱数据拟合分析的技能，以确定N末端的探针到探针距离和障碍 mybp-c。第三阶段将通过提供结构的分子细节来促进目标1和AIM 2 肌动蛋白 - mybp-C复合物的动力学，添加的肌钙蛋白，肌球蛋白和肌球蛋白到模拟生理 肌肉中心脏细丝的条件。候选人将在模型系统中系统地构建 从肌动蛋白结合到薄丝结合的MYBP-C的复杂性，具有低（舒张）或高（收缩）激活剂Ca2+， 心脏肌球蛋白结合后，在肌丝水平上提供关键见解 了解肌肉细胞中的基本机制。 MYBP-C调节的光谱研究 细丝将确定蛋白质相互作用和结构动力学。该项目基于基本 生物物理学机制，但MYBP-C已成为心脏病的治疗靶标。那是我们的工作 使用我们独特的光谱方法为药物疗法开发筛查的基础。