Structural Dynamics of Cardiac Myosin Binding Protein-C

描述(申请人提供)：这是一个名为“心肌肌球蛋白结合蛋白-C的结构动力学”的项目的重新提交。这个项目的主要目标是为我的职业生涯做好准备，成为一名肌肉分子生物物理学的独立研究员，特别是心脏。我在心脏和骨骼肌生理学方面有很强的博士前背景，使用肌肉纤维力学和X射线衍射法，通过接受光谱探测技术方面的培训，我得到了加强，这是导师实验室的独特优势。指导计划现在专注于一个研究项目，该项目综合了我以前的经验，并提出了关于蛋白质结构动力学和磷酸化在肌肉功能中的作用的基本问题。肌球蛋白结合蛋白-C(CPRO)在肥厚型心肌病(HCM)和心力衰竭的心功能调节和收缩功能缺陷中起重要作用。我的项目还侧重于了解和缓解HCM疾病的机制，因为有必要解决该蛋白与肌球蛋白和肌动蛋白复合体的结构和动力学以及CPRO相关HCM疾病发生和发展中随之而来的收缩功能障碍等基本问题。这次重新提交有三个具体目的：(1)使用与肌球蛋白或肌动蛋白特异性结合的自旋标记的电子顺磁共振(EPR)，准确地测量CPRO基因消融对几种人肥厚性心肌病小鼠模型皮肤心肌纤维结构动力学的影响。(2)用Aim 1的方法定量测定依赖PKA的CPRO磷酸化对肌球蛋白和肌动蛋白结构动力学的影响。评估交换纤维和完整心脏的基线和磷酸化收缩性能。(3)利用偶极电子-电子共振(DER)技术并将该方法扩展到肌球蛋白或肌动蛋白溶液中CPRO的探针，定量测定结合、磷酸化和HCM突变对CPRO特定区域结构动力学的影响。我将利用这些机制细节为CPRO的小分子效应器的高通量药物检测提供基础，作为治疗肥厚性心肌病和心力衰竭的潜在疗法。我最近发表在PNAS上的文章揭示了有关CPRO及其磷酸化对标记的肌动蛋白细丝结构动力学的影响的令人兴奋的新信息，这些结果将为所有三个目标提供信息。所有这三个目标都已经确定了可行性，下面的提案描述了在持续监督的研究经验下实现我的目标所需的工作--在两年内发表几篇影响较大的同行评议论文，增强我在这些研究领域的专业知识，并准备成立我自己的独立实验室。

DESCRIPTION (provided by applicant): This is a resubmission of a project entitled "Structural Dynamics of Cardiac Myosin Binding Protein-C." The primary goal of this project is to prepare me for a career as an independent investigator in molecular biophysics of muscle, with particular emphasis on the heart. My strong predoctoral background in heart and skeletal muscle physiology, using methods of muscle fiber mechanics and X-ray diffraction, has been augmented by training in spectroscopic probe techniques, which are particular strengths of the mentor's laboratory. The mentoring program now focuses on a research project that synthesizes my previous experiences and asks fundamental questions about the role of protein structural dynamics and phosphorylation in the function of muscle. Myosin binding protein-C (CPro) plays a major role in the modulation of cardiac function and in deficits of contractile function in hypertrophic cardiomyopathy (HCM) and heart failure. My Project also focuses on understanding and alleviating HCM disease mechanisms, as it is necessary to resolve fundamental questions about the structure and dynamics of this protein's complexes with myosin and actin as well as the contractile dysfunction that ensues in the CPro-associated HCM disease onset and progression. There are three specific aims of this Resubmission: (1) Use electron paramagnetic resonance (EPR) of spin labels specifically bound to myosin or actin, to measure accurately the effects of genetic ablation of CPro on structural dynamics in skinned cardiac muscle fibers, from several mouse models of human HCM. (2) Use the approach of Aim 1 to determine quantitatively the effects of PKA-dependent phosphorylation of CPro on myosin and actin structural dynamics. Assess baseline and phosphorylated contractile performance in exchanged fibers and intact hearts. (3) Use dipolar electron-electron resonance (DEER) and extend this method to FRET with probes on CPro in solution with myosin or actin, to determine quantitatively the effects of binding, phosphorylation, and HCM mutation on structural dynamics of specific regions of CPro. I will use these mechanistic details to provide the bases for a high-throughput drug assay for small molecule effectors of CPro as potential therapy for HCM and heart failure. My recent PNAS publication revealed exciting new information about the effects of CPro and its phosphorylation on the structural dynamics of labeled actin filaments, and these results will inform all three aims. Feasibility has been established for all three aims, and the proposal below describes the work needed to achieve my goals under continued supervised research experience -- to publish several high-impact peer- reviewed papers within two years, augment my expertise in these areas of research, and prepare to start my own independent lab.