Diversity Supplement to Skeletal Myosin-Binding Protein C Regulation and Structural Dynamics

骨骼肌球蛋白结合蛋白 C 调节和结构动力学的多样性补充

• 批准号: 10824055
• 负责人: Brett A Colson
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10824055
• 关键词: Acceleration; Actins; Actomyosin; Address; Affect; Anisotropy; Arthrogryposis; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Biophysical Process; Biophysics; C2 Domain; Cardiac Myosins; Complex; Contracture; Cyclic AMP-Dependent Protein Kinases; DAG/PE-Binding Domain; Deceleration; Detection; Development; Disease; Distal; Drug Screening; Equilibrium; Filament; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Frequencies; Gel; Genes; Genetic; Goals; Health; Human; Hypertrophic Cardiomyopathy; In Vitro; Joints; Kinetics; Knowledge; Label; Learning; Live Birth; Location; Lower Extremity; Measures; Mediating; Medical; Methods; Microfilaments; Molecular; Molecular Conformation; Monitor; Muscle; Muscle Cells; Muscle Proteins; Muscle Weakness; Muscle function; Mutation; Myocardial; Myopathy; Myosin ATPase; N-terminal; Outcome; Parents; Pathogenicity; Pathologic; Performance; Pharmaceutical Preparations; Pharmacotherapy; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Positioning Attribute; Protein Dynamics; Protein Isoforms; Proteins; Publishing; RNA Splicing; Recombinants; Regulation; Reporter; Reporting; Research; Research Project Grants; Research Training; Resolution; Role; Sarcomeres; Scientist; Site; Skeletal Muscle; Slide; Spectrum Analysis; Stains; Structure; Technology; Testing; Therapeutic; Thick Filament; Thin Filament; Time; Training; Tropomyosin; Troponin; Upper Extremity; Variant; Visualization; Work; biophysical tools; career development; design; effective therapy; heart function; improved; innovation; insight; mutant; myosin-binding protein C; nanometer; next generation; novel; novel therapeutics; parent grant; parent project; phosphorescence; prevent; protein complex; protein structure; reconstitution; skeletal; skills; spectroscopic data; spectroscopic survey; therapeutic target; time use; tool

Title of project: DIVERSITY SUPPLEMENT TO SKELETAL MYOSIN-BINDING PROTEIN C REGULATION & STRUCTURAL DYNAMICS. Summary of the diversity supplement to the parent project. Skeletal 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 distal arthrogryposis (DA) and the role of phosphorylation and DA mutations is not known. Our goal is to understand the molecular biophysics of muscle 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 skeletal 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, DA mutations, and MyBP-C drugs. 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 DA mutations influence N-terminal and central domain skeletal MyBP-C binding with actin and/or myosin in a dynamic equilibrium to modulate contraction. Related to the parent grant, the first period of the diversity supplement focuses on using spectroscopic approaches to accurately measure the structural dynamics within, and adjacent to, the Pro/Ala-rich linker (P/A) of purified skeletal MyBP-C fragment C1-C7, primarily by measuring nanometer distances and molecular disorder. Major emphasis is placed on detection of conformational changes (structure) within and nearby MyBP-C’s P/A due to phosphorylation, DA mutation, actin or myosin binding (function), and drugs. By including the location of probes in P/A and in adjacent C1 and C2 domains, the Candidate will measure structural changes. Fluorescently-labeled MyBP-C will be prepared to acquire fluorescence lifetime using time-resolved methods. Human splice variants containing and missing the phosphorylation site in long and short forms in P/A will be evaluated. 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 and central domain MyBP-C. The third period will provide molecular details of the structural dynamics upon phosphorylation of P/A in long form sMyBP-C and actin- or myosin- MyBP-C complexes. The Candidate will systematically build in model system complexity, from unbound to actin/myosin-bound MyBP-C, upon phosphorylation. Spectroscopic study of sMyBP-C regulation will determine protein interactions and structural dynamics, providing key insights at the myofilament level to be applied for understanding fundamental mechanisms in the muscle cell. This project is grounded in fundamental biophysics mechanisms, but MyBP-C has emerged as a therapeutic target for skeletal muscle disease. Thus, of our work lays a foundation for testing identified drugs and development of screens for drug therapies using our unique spectroscopic approaches.

项目标题：骨骼肌球蛋白结合蛋白C调节的多样性补充 结构动力学。 母公司项目多样性补充的摘要。 骨骼肌球蛋白结合蛋白C（MYBP-C）在心脏功能的调节中起主要作用 磷酸化并导致由于远端关节炎（DA）中MYBP-C突变引起的收缩功能的定义 尚不清楚磷酸化和DA突变的作用。我们的目标是了解分子 肌肉的生物物理学并训练下一代肌肉生物物理学家，包括潜水员学员。这 家长研究项目和多样性补充提出有关蛋白质相互作用作用的基本问题 和调节骨骼肌功能的结构动力学。为了深入了解结构的相关性 - MYBP-C机制涉及物理和病理环境中的功能，我们将探测肌动蛋白 这些蛋白质中这些蛋白质中的肌球蛋白 - mybp-c复合物具有不同的结合，磷酸化，DA突变和 MYBP-C药物。我们的核心技术是位于现场的光谱法，用于纯化的MYBP-C和肌动蛋白/肌球蛋白 细丝。我们将在现场定向的标签和光谱中应用创新的互补方法 相关的蛋白质结合，结构动力学和功能。我们将测试中心假设 磷酸化和DA突变影响N端和中央域骨骼MYBP-C与肌动蛋白结合 和/或肌球蛋白在动态上等效地调节收缩。与父母赠款有关的第一阶段 多样性补充剂的重点是使用光谱方法准确测量结构 纯化的骨骼MYBP-C片段C1-C7的动力学和与富含/ALA的接头（P/A）， 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，DA突变，肌动蛋白 或肌球蛋白结合（功能）和药物。通过在P/A中以及相邻的C1和C2中包括问题的位置 域，候选人将测量结构变化。荧光标记的MYBP-C将准备好 使用时间分辨方法获取荧光寿命。人类剪接变体包含和缺少 将评估P/A中长形式和短形式的磷酸化位点。在第二阶段，候选人将 学习光谱数据拟合分析的新技能，以确定探针到探针距离和n- 终端和中央域mybp-c。第三阶段将提供结构动力学的分子细节 长期形式的p/a磷酸化后，smybp-c和肌动蛋白或肌动蛋白mybp-c络合物。候选人 将在模型系统的复杂性中系统地构建，从未结合到肌动蛋白/肌球蛋白结合MyBP-C， 磷酸化。 SMYBP-C调节的光谱研究将确定蛋白质相互作用和结构 动态，提供在肌丝层的关键见解，以理解基本 肌肉细胞中的机制。该项目基于基本生物物理学机制，但MYBP-C 已成为骨骼肌疾病的治疗靶标。我们的工作为测试奠定了基础 使用我们独特的光谱方法确定了药物和用于药物疗法的筛查。