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

项目名称：骨骼肌球蛋白结合蛋白C调节的多样性补充 结构动力学。 对母项目的多样性补充进行总结。 骨骼肌球蛋白结合蛋白C(MyBP-C)在心脏功能调节中起重要作用。 远端关节紊乱病(DA)中MyBP-C突变引起的磷酸化和收缩功能缺陷 而磷酸化和DA突变的作用尚不清楚。我们的目标是了解分子 肌肉生物物理学和培养下一代肌肉生物物理学家，包括不同的受训人员。这个 父母研究项目和多样性补充提出了关于蛋白质相互作用的基本问题 以及调节骨骼肌功能的结构动力学。为了洞察结构之间的关联-- 在生理和病理背景下，我们将探讨肌动蛋白-C的作用机制。 这些蛋白在溶液中的肌球蛋白-MyBP-C复合体具有不同的结合、磷酸化、DA突变和 MyBP-C类药物。我们的核心技术是定点光谱，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分析方面应用创新的互补方法 将蛋白质结合、结构动力学和功能联系起来。我们将检验核心假设，即 磷酸化和DA突变影响肌动蛋白N末端和中央结构域骨架MyBP-C的结合 和/或肌球蛋白处于动态平衡以调节收缩。与父母助学金有关的第一期 多样性补充侧重于使用光谱方法来准确测量结构 纯化的骨骼MyBP-C片段C1-C7的富含Pro/Ala的连接体(P/A)内及其附近的动力学， 主要是通过测量纳米距离和分子无序。主要的重点是检测 由磷酸化、DA突变、肌动蛋白引起的MyBP-C的P/A及其附近的构象变化(结构) 或肌球蛋白结合(功能)，以及药物。通过在P/A以及相邻的C1和C2中包括探头的位置 对于域名，候选者将衡量结构变化。荧光标记的MyBP-C将被制备成 用时间分辨的方法获得荧光寿命。人类剪接变异体包含和缺失 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中的新技能，以确定N-N中的探头到探头的距离和无序 末端和中央结构域MyBP-C。第三期将提供结构动力学的分子细节。 当P/A磷酸化时，形成长形式的sMyBP-C和肌动蛋白-或肌球蛋白-MyBP-C复合体。候选人 将系统地构建模型系统复杂性，从非结合到肌动蛋白/肌球蛋白结合的MyBP-C， 磷酸化。对sMyBP-C调控的光谱研究将确定蛋白质的相互作用和结构 动力学，提供肌丝水平的关键见解，用于理解基础知识 肌肉细胞中的机制。该项目基于基本的生物物理机制，但MyBP-C 已经成为骨骼肌疾病的治疗靶点。因此，我们的工作为测试奠定了基础 使用我们独特的光谱方法，识别药物并开发药物治疗筛查。

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.