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

基本信息

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
项目类别：
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

项目摘要

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 调节的多样性补充结构动力学。对父项目的多样性补充的摘要。骨骼肌球蛋白结合蛋白 C (MyBP-C) 通过其在心脏功能的调节中发挥着重要作用远端关节弯曲 (DA) 中 MyBP-C 突变导致磷酸化并导致收缩功能缺陷磷酸化和 DA 突变的作用尚不清楚，我们的目标是了解分子机制。肌肉生物物理学并培训下一代肌肉生物物理学家，包括不同的受训者。家长研究项目和多样性补充提出了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以深入了解结构的相关性。在生理和病理环境中参与 MyBP-C 机制的功能，我们将探讨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 突变和 MyBP-C 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。我们将在定点标记和光谱学中应用创新的补充方法。我们将检验中心假设：蛋白质的结合、结构动力学和功能。磷酸化和 DA 突变影响 N 端和中心结构域骨骼 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与母体授予相关的收缩。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架 MyBP-C 片段 C1-C7 的富含 Pro/Ala 的接头 (P/A) 内及其附近的动态，主要通过测量纳米距离和分子紊乱进行检测。由于磷酸化、DA 突变、肌动蛋白，MyBP-C 的 P/A 内部和附近的构象变化（结构）或肌球蛋白结合（功能），以及药物通过包括 P/A 以及相邻 C1 和 C2 中的探针位置。域，候选人将准备测量荧光标记的 MyBP-C 的结构变化。使用时间分辨方法获得包含和缺失的人类剪接变体的荧光寿命。在第二阶段，候选人将评估 P/A 中长形式和短形式的磷酸化位点。学习光谱数据拟合分析的新技能，以确定 N- 中的探针间距离和无序度末端和中心结构域 MyBP-C。第三阶段将提供结构动力学的分子细节。长形式 sMyBP-C 和肌动蛋白-或肌球蛋白-MyBP-C 复合物中 P/A 的磷酸化将系统地构建模型系统复杂性，从未结合到肌动蛋白/肌球蛋白结合的 MyBP-C， sMyBP-C 调节的光谱研究将确定蛋白质相互作用和结构。动力学，提供肌丝水平的关键见解，可用于理解基本原理该项目以基本生物物理学机制为基础，但 MyBP-C 已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确定和开发药物治疗筛选。