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

基本信息

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
项目类别：
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

项目摘要

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.

项目名称：心脏肌球蛋白结合结构动力学的多样性补充蛋白质 C 调节。肌球蛋白结合蛋白 C (MyBP-C) 通过其在心脏功能的调节中发挥着重要作用磷酸化并导致肥大肌中 MyBP-C 突变导致收缩功能缺陷心肌病（HCM）和心力衰竭中磷酸化的降低我们的目标是了解分子机制。肌肉的生物物理学，特别是心脏，并训练下一代肌肉生物物理学家，包括不同的受训者，母公司的研究项目和多样性补充要求。关于蛋白质相互作用和调节功能的结构动力学的作用的基本问题深入了解 MyBP-C 机制中涉及的结构与功能的相关性。生理和病理环境中，我们将探究这些蛋白质的肌动蛋白-肌球蛋白-MyBP-C 复合物具有不同结合、磷酸化和 HCM 突变的解决方案我们的核心技术是定点技术。光谱，应用于纯化的 MyBP-C 和肌动蛋白/肌球蛋白丝，我们将应用创新的互补技术。定点标记和光谱学方法与蛋白质相关结合、结构动力学和功能。我们将测试 N 端 MyBP-C 的磷酸化和 HCM 突变改变的中心假设 MyBP-C 单独和与肌动蛋白和肌球蛋白相互作用时具有重要的功能结构特性。家长拨款目标 1，多样性补充的第一阶段重点是使用光谱方法准确测量纯化的 MyBP-C M 结构域内发生磷酸化的结构动力学，主要通过测量纳米距离和分子紊乱进行检测。由于磷酸化、HCM 突变和肌动蛋白，MyBP-C 的 M 结构域内的构象变化（结构）或肌球蛋白结合（功能）通过在 M 结构域中包含探针的位置，候选人将进行测量。将准备通过我们的计算模拟预测的结构变化。使用时间分辨方法获取荧光寿命在第二阶段，考生将学习新的知识。光谱数据拟合分析技能，以确定探针间距离和 N 端紊乱 MyBP-C。第三阶段将通过提供结构的分子细节对目标 1 和目标 2 做出贡献。添加肌钙蛋白、原肌球蛋白和肌球蛋白以模拟生理学的肌动蛋白-MyBP-C 复合物的动力学考生将系统地建立模型系统。复杂性，从肌动蛋白结合到细丝结合 MyBP-C，具有低（舒张期）或高（收缩期）激活剂 Ca2+，并在与心肌肌球蛋白结合后，提供肌丝水平的关键见解以应用于了解肌肉细胞中 MyBP-C 调节的基本机制。细丝将决定蛋白质相互作用和结构动力学该项目以基础为基础。生物物理学机制，但 MyBP-C 已成为心脏病的治疗靶点。为使用我们独特的光谱方法开发药物治疗筛选奠定了基础。