cMyBP-C and Native Thick Filament Structure

肌球蛋白结合蛋白C （MyBP-C）是脊椎动物骨骼肌和心肌粗纤维的组成部分。心肌同种异构体cMyBP-C的磷酸化在响应p-肾上腺素能刺激的心脏收缩性调节中起关键作用。cMyBP-C突变已被证明是心脏病家族性肥厚性心肌病的主要原因。我们的长期目标是了解cMyBP-C函数的结构基础。在这个项目中，电子显微镜和图像处理将被用来阐明分子的结构，它在肌节中的组织，它与细丝的相互作用，以及当它被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端片段（在Core C中产生），野生型和转基因心脏（Core C）的天然粗丝，以及完整的肌肉。将讨论三个具体目标。(1) cMyBPC在分子、粗丝和肌体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、沿着纤维表面延伸或从纤维表面伸出以与邻近的纤维相互作用，以及它如何影响肌凝蛋白头部组织。(2) cMyBP-C调控肌动蛋白-肌球蛋白相互作用的结构基础是什么？我们将确定cMyBP-C是否与肌凝蛋白头竞争肌动蛋白结合，以及在高或低Ca[2+]水平下，cMyBP-C是否影响原肌凝蛋白在薄纤维上的位置。(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、粗丝结构（如头部构象）及其与细丝的相互作用。这些目标将通过使用阴性染色，冷冻电镜，抗体标记和肌肉切片方法，结合单颗粒，螺旋和层析三维重建技术来实现。结果将与，和相关

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.