Thin Filaments and Muscle Regulation

细丝和肌肉调节

• 批准号: 8605902
• 负责人: WILLIAM J LEHMAN
• 金额: $ 40.11万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8605902
• 关键词: Actin-Binding Protein; Actins; Address; Architecture; Asthma; Attention; Binding; Binding Proteins; Binding Sites; Blood Vessels; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular system; Chemicals; Computational Biology; Coupled; Data; Development; Disease; Electrons; Filament; Functional disorder; Goals; Hypertension; Image Analysis; Inherited; Lead; Link; Lung; Lung diseases; Microfilaments; Molecular; Molecular Structure; Movement; Muscle; Muscle Contraction; Mutation; Myocardium; Myosin ATPase; Participant; Process; Protein Dynamics; Proteins; Protocols documentation; Regulation; Resistance; Resolution; Respiration Disorders; Skeletal Muscle; Smooth Muscle; Structure; System; Techniques; Testing; Thin Filament; Tropomyosin; Troponin; Work; base; computerized tools; design; genetic regulatory protein; innovation; link protein; molecular dynamics; mutant; myosin-binding protein C; particle; reconstruction; structural biology; therapeutic target

Thin filament-linked actin-binding proteins control both actomyosln-based muscle contraction and cytoskeletal formation. In order to understand the normal control of muscle contraction, it is crucial to characterize the structural interactions of thin filament regulatory proteins that influence actin-mvosin association. Using structural analysis, we will examine the architecture of cardiac and skeletal muscle thin filaments at a fundamental level and determine the changing interactions of thin filament-linked proteins that regulate muscle activity. Our principal objective therefore is to solve the atomic structure of the entire thin filament in relaxed and in active muscle. We use state-of-the-art electron microscopv and electron tomographv. coupled with image analysis, 3D reconstruction (3DEM) and computational ehemistrv to establish the macromolecular structure of actin-binding proteins on thin filament actin and thus demarcate molecular contacts of binding proteins with actin at near atomic resolution. Using these techniques: (1) We aim to detennine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca^^-binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (a) we will describe the complete atomic structure of tropomyosin and troponintropomyosin on thin filaments by generating single particle and electron tomographic reconstructions at increasingly high resolution and by fitting atomic resolution crystal structures of actin and regulatory proteins into the reconstruction volumes; (b) we will further refine these atomic structures using computational tools to maximize chemical interactions between regulatory proteins and actin; (c) we will use Molecular Dynamics protocols to assess regulatory protein dynamics and likely transitions between regulatory states. (2) Using this same approach, we will test the hypothesis that mutant cardiac troponin and tropomyosin perturb muscle regulation by causing imbalanced protein interactions that alter the regulatory state of thin filaments, and we will determine the underlying structural reasons for such perturbations. (3) We will test the hypothesis that the short molecular overlap domain between adjacent tropomyosins, needed for end-to-end tropomyosin linkage, is vital for tropomyosin strand formation on filaments, and again assess the impact of mutants on this structure. (4) We will complete efforts to test the hypothesis that Myosin-Binding Protein C forms regular and periodic links to thin filaments, which are likely to modulate striated contractile activity.

细丝连接的肌动蛋白结合蛋白控制基于肌动蛋白的肌肉收缩和 细胞骨架形成。为了了解肌肉收缩的正常控制，至关重要的是 表征影响肌动蛋白-肌动蛋白的细丝调节蛋白的结构相互作用 协会。利用结构分析，我们将研究心肌和骨骼肌的结构。 细丝在基态水平上的变化和决定细丝连接相互作用的变化 调节肌肉活动的蛋白质。因此，我们的主要目标是解决原子 整个细丝的结构在放松和活跃的肌肉中。我们使用最先进的 电子显微镜和电子断层摄影术。结合图像分析、三维重建(3DEM) 以及建立肌动蛋白结合蛋白的大分子结构的计算方法 细丝肌动蛋白，从而划分结合蛋白与肌动蛋白在近原子的分子接触 决议。利用这些技术：(1)我们的目标是确定肌钙蛋白-原肌球蛋白的结构基础 通过分析原肌球蛋白和肌钙蛋白的相互作用来调节心肌和骨骼肌的活动 在细丝上，由肌钙蛋白与肌球蛋白的结合和肌球蛋白与肌动蛋白的交叉桥结合控制。 为了实现这一目标，(A)我们将描述原肌球蛋白和肌钙蛋白的完整原子结构 通过产生单粒子和电子层析重建来研究细丝 肌动蛋白和调节蛋白的分辨率越来越高，并通过拟合原子分辨率晶体结构 进入重建卷；(B)我们将使用计算工具进一步精炼这些原子结构 以最大化调节蛋白质和肌动蛋白之间的化学相互作用；(C)我们将使用分子 评估调控蛋白质动力学和可能在调控状态之间转换的动力学协议。 (2)使用同样的方法，我们将检验突变的心肌肌钙蛋白和原肌球蛋白 通过引起不平衡的蛋白质相互作用来扰乱肌肉的调节，从而改变瘦的调节状态 细丝，我们将确定这种扰动的潜在结构性原因。(3)我们将测试 假设相邻原肌球蛋白之间的短分子重叠结构域是端到端所需的 原肌球蛋白连接，对于原肌球蛋白链在细丝上的形成至关重要，并再次评估了 这个结构上的变种。(4)我们将完成检验肌球蛋白结合蛋白C假设的努力 形成规则的和周期性的连接到细丝，这可能调制横纹收缩活动。