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.

项目名称：骨骼肌球蛋白结合蛋白C调节的多样性 结构化学。 父项目的多样性补充摘要。 心肌肌球蛋白结合蛋白C（MyBP-C）通过其特异性结合在心功能调节中起重要作用。 在远端关节弯曲（DA）中由于MyBP-C突变导致的磷酸化和收缩功能缺陷 磷酸化和DA突变的作用尚不清楚。我们的目标是了解 肌肉生物物理学和培养下一代肌肉生物物理学家，包括不同的学员。的 父母研究项目和多样性补充问蛋白质相互作用的作用的基本问题 以及调节骨骼肌功能的结构动力学。为了深入了解结构的相互关系- 在生理和病理环境中参与MyBP-C机制的功能，我们将探测肌动蛋白- 这些蛋白质在溶液中的肌球蛋白-MyBP-C复合物具有不同的结合、磷酸化、DA突变， MyBP-C药物。我们的核心技术是定点光谱法，应用于纯化的MyBP-C和肌动蛋白/肌球蛋白 细丝。我们将在定点标记和光谱学中应用创新的互补方法， 将蛋白质结合、结构动力学和功能关联起来。我们将检验中心假设， 磷酸化和DA突变影响N-末端和中心结构域骨架MyBP-C与肌动蛋白的结合 和/或肌球蛋白处于动态平衡以调节收缩。与父母补助金有关， 多样性补充侧重于使用光谱方法来准确测量结构 纯化的MyBP-C骨架片段C1-C7的富含Pro/Ala的接头（P/A）内和附近的动力学， 主要是通过测量纳米距离和分子无序度。主要重点放在检测 由于磷酸化、DA突变、肌动蛋白 或肌球蛋白结合（功能）和药物。通过包括P/A和相邻C1和C2中探头的位置 域，候选人将衡量结构变化。将制备标记的MyBP-C， 使用时间分辨方法获得荧光寿命。含有和缺失 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N- 末端和中心结构域MyBP-C。第三节课将提供结构动力学的分子细节 在长形式sMyBP-C和肌动蛋白-或肌球蛋白- MyBP-C复合物中的P/A磷酸化。候选 将系统地建立模型系统的复杂性，从未结合到肌动蛋白/肌球蛋白结合的MyBP-C， 磷酸化sMyBP-C调控的光谱研究将确定蛋白质相互作用和结构 动力学，在肌丝水平上提供关键的见解，用于理解基本的 肌肉细胞中的机制。该项目基于基本的生物物理学机制，但MyBP-C 已经成为骨骼肌疾病的治疗靶点。从而为我们的工作奠定了测试的基础 利用我们独特的光谱方法鉴定药物和开发药物治疗筛选。