Structural Dynamics of Cardiac Myosin Binding Protein-C

描述（由申请人提供）：这是一个题为“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，作为肌肉分子生物物理学的独立研究者，特别强调心脏。我在心脏和骨骼肌生理学方面的博士前背景很强，使用肌纤维力学和X射线衍射的方法，通过光谱探针技术的培训得到了增强，这是导师实验室的特别优势。导师计划现在专注于一个研究项目，综合我以前的经验，并询问有关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）和心力衰竭的心脏功能调节和收缩功能缺陷中起重要作用。我的项目还侧重于理解和缓解HCM疾病机制，因为有必要解决有关这种蛋白质与肌球蛋白和肌动蛋白的复合物的结构和动力学的基本问题，以及在CPro相关的HCM疾病发作和进展中引起的收缩功能障碍。 本重新提交有三个具体的目的：（1）使用电子顺磁共振（EPR）的自旋标记特异性结合到肌球蛋白或肌动蛋白，以准确测量的影响，基因消融CPro对皮肤的心肌纤维的结构动力学，从几个小鼠模型的人HCM。(2)采用Aim 1的方法定量测定PKA依赖的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)使用偶极电子-电子共振（DEER），并将此方法扩展到FRET与肌球蛋白或肌动蛋白溶液中的CPro上的探针，以定量确定结合，磷酸化和HCM突变对CPro的特定区域的结构动力学的影响。我将使用这些机制的细节，为高通量的药物检测CPro的小分子效应物作为HCM和心力衰竭的潜在治疗提供基础。 我最近的PNAS出版物揭示了关于CPro及其磷酸化对标记肌动蛋白丝结构动力学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。所有三个目标的可行性都已经确定，下面的建议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-在两年内发表几篇高影响力的同行评审论文，增强我在这些研究领域的专业知识，并准备开始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.