cMyBP-C and Native Thick Filament Structure

cMyBP-C 和天然粗丝结构

• 批准号: 8215308
• 负责人: ROGER W CRAIG
• 金额: $ 46.86万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8215308
• 关键词: Actins; Actomyosin; Address; Adrenergic Agents; Antibodies; Binding; Biophysics; C-terminal; Cardiac; Cardiac Myosins; 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; Microfilaments; Modeling; Molecular; Molecular Conformation; Muscle; Mutation; Myocardium; Myosin ATPase; N-terminal; Negative Staining; Phosphorylation; Physiological; Play; Positioning Attribute; Protein Isoforms; Proteins; Pump; Regulation; Resolution; Role; Running; Sarcomeres; Sectioning technique; Site; Skeletal Muscle; 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

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脏肌肉厚细丝的组成部分。心脏同工型CMYBP-C的磷酸化在调节心脏收缩率时起着关键作用，以响应P-肾上腺素能刺激。 CMYBP-C中的突变已被证明是心脏病，家族性心肌病的主要原因。我们的长期目标是了解CMYBP-C功能的结构基础。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，与细丝薄丝的相互作用以及磷酸化时发生的变化。实验将利用表达的突变体和野生型CMYBP-C分子和N末端片段（核心C），来自野生型和转基因心脏（核心C）的天然厚细丝（核心C）以及完整的肌肉。将解决三个具体目标。 （1）如何在分子，较厚的细丝和肉瘤水平上组织CMYBPC？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，无论它是环绕，延伸还是从细丝表面延伸以与邻近的细丝相互作用，以及它如何影响肌球蛋白头组织。 （2）CMYBP-C对肌动蛋白肌球蛋白相互作用的调节的结构基础是什么？我们将确定CMYBP-C是否与肌球蛋白头竞争肌动蛋白结合，以及它是否影响肌动蛋白在高或低Ca [2+]水平上的薄膜上的位置。 （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的柔韧性，较厚的细丝结构（例如头部构象）以及与细丝的相互作用。这些目标将使用阴性污渍，冷冻EM，抗体标记和肌肉切片方法，结合单个粒子，螺旋和断层扫描3D重新构造技术。结果将与 在结构上是平行的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）。该项目将提供有关CMYBP-C函数心脏中的结构机制的新见解。