cMyBP-C and Native Thick Filament Structure

cMyBP-C 和天然粗丝结构

• 批准号: 7789873
• 负责人: ROGER W CRAIG
• 金额: $ 48.08万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7789873
• 关键词: Actins; Actomyosin; Address; Adrenergic Agents; Antibodies; Arts; Binding; Biophysics; C-terminal; Cardiac; Cryoelectron Microscopy; Data; Defect; Electron Microscopy; F-Actin; Familial Hypertrophic Cardiomyopathy; Filament; Foundations; Goals; Head; Heart; Heart Diseases; Hypertrophic Cardiomyopathy; Image; In Vitro; Individual; Inherited; Knowledge; Label; Modeling; Molecular; Molecular Conformation; Muscle; Mutation; Myocardium; Myosin ATPase; N-terminal; Negative Staining; Phosphorylation; Physiological; Play; Positioning Attribute; Protein Isoforms; Proteins; Pump; Resolution; Role; Running; Sarcomeres; Sectioning technique; Site; Solid; Structural Models; Structure; Surface; Techniques; Testing; Thick; Thick Filament; Thin Filament; Transgenic Organisms; Tropomyosin; Vertebral column; adrenergic; base; citrate carrier; flexibility; image processing; in vivo; insight; mutant; myosin-binding protein C; particle; research study; response; single molecule; skeletal

Myosin-binding protein C (MyBP-C) is a component of the thick filaments of vertebrate skeletal and cardiac muscle. Phosphorylation of the cardiac isoform, cMyBP-C, plays a key role in modulating cardiac contractility in response to p-adrenergic stimulation. Mutations in cMyBP-C have been shown to be a prime cause of the cardiac disease, familial hypertrophic cardiomyopathy. Our long term goal is to understand the structural basis of cMyBP-C function. In this project electron microscopy and image processing will be used to elucidate the structure of the molecule, its organization in the sarcomere, its interaction with thin filaments, and the changes that occur when it is phosphorylated. Experiments will make use of expressed mutant and wild type cMyBP-C molecules and N-terminal fragments (produced in Core C), native thick filaments from wild type and transgenic hearts (Core C), and intact muscle. Three specific aims will be addressed. (1) How is cMyBPC organized at the molecular, thick filament and sarcomeric level? We will determine whether the MyBP-C molecule has specific structural features required to carry out its function, whether it wraps around, extends along, or projects away from the filament surface to interact with neighboring filaments, and how it influences myosin head organization. (2) What is the structural basis of cMyBP-C's modulation of actin-myosin interaction? We will determine whether cMyBP-C competes with myosin heads for actin binding, and whether it influences the position of tropomyosin on thin fliaments at high or low Ca[2+] levels. (3) What are the structural effects of cMyBP-C phosphorylation? We will determine whether phosphorylation alters cMyBP-C flexibility, thick filament structure (e.g. head conformation), and its interaction with thin filaments. These goals will be achieved using negative stain, cryo-EM, antibody labeling and muscle sectioning approaches, combined with single particle, helical and tomographic 3D reconstrucfion techniques. Results will be correlated with, and structurally underpin, parallel single molecule biophysics experiments (Project 2) and whole heart functional data (Project 3, Core B). The project will provide new insights into the structural mechanisms by which cMyBP-C funcfions in the heart.

肌球蛋白结合蛋白 C (MyBP-C) 是脊椎动物骨骼和心肌粗丝的组成部分。心脏异构体 cMyBP-C 的磷酸化在调节 p-肾上腺素能刺激反应的心肌收缩力中发挥着关键作用。 cMyBP-C 突变已被证明是心脏病、家族性肥厚型心肌病的主要原因。我们的长期目标是了解 cMyBP-C 函数的结构基础。在该项目中，电子显微镜和图像处理将用于阐明分子的结构、其在肌节中的组织、其与细丝的相互作用以及其磷酸化时发生的变化。实验将利用表达的突变型和野生型 cMyBP-C 分子和 N 末端片段（在 Core C 中产生）、来自野生型和转基因心脏 (Core C) 的天然粗丝以及完整的肌肉。将解决三个具体目标。 (1) cMyBPC 在分子、粗丝和肌节水平上是如何组织的？我们将确定 MyBP-C 分子是否具有执行其功能所需的特定结构特征，它是否环绕、沿着细丝表面延伸或远离细丝表面突出以与邻近的细丝相互作用，以及它如何影响肌球蛋白头部组织。 (2) cMyBP-C 调节肌动蛋白-肌球蛋白相互作用的结构基础是什么？我们将确定 cMyBP-C 是否与肌球蛋白头竞争肌动蛋白结合，以及它是否在高或低 Ca[2+] 水平下影响原肌球蛋白在细丝上的位置。 (3) cMyBP-C磷酸化的结构效应是什么？我们将确定磷酸化是否会改变 cMyBP-C 的灵活性、粗丝结构（例如头部构象）及其与细丝的相互作用。这些目标将通过使用负染色、冷冻电镜、抗体标记和肌肉切片方法，结合单颗粒、螺旋和断层扫描 3D 重建技术来实现。结果将与相关，并且 从结构上支持并行单分子生物物理学实验（项目 2）和整个心脏功能数据（项目 3，核心 B）。该项目将为 cMyBP-C 在心脏中发挥作用的结构机制提供新的见解。