Structural Dynamics of Cardiac Myosin Binding Protein-C

心肌肌球蛋白结合蛋白-C 的结构动力学

• 批准号: 8791218
• 负责人: Brett A Colson
• 金额: $ 13.52万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791218
• 关键词: ATP phosphohydrolase; Ablation; Actins; Actomyosin; Affect; Affinity; Area; Binding; Binding Proteins; Biochemistry; Biological Assay; Biological Models; Biophysics; Cardiac; Cardiac Myosins; Clinical; Consult; Cyclic AMP-Dependent Protein Kinases; Data Analyses; Development; Development Plans; Disease; Disease Progression; Drug Compounding; Electron Spin Resonance Spectroscopy; Electrons; Engineering; Fiber; Fluorescence Resonance Energy Transfer; Frequencies; Functional disorder; Genetic; Genetic Anticipation; Goals; Hand; Health; Heart; Heart Diseases; Heart failure; Human; Hypertrophic Cardiomyopathy; Knock-out; Knockout Mice; Label; Laboratories; Libraries; Measures; Mechanics; Mediating; Mentors; Methods; Microfilaments; Molecular; Molecular Biology; Molecular Conformation; Monitor; Muscle; Muscle Contraction; Muscle Fibers; Muscle Proteins; Muscle function; Mutation; Myocardium; Myosin ATPase; Myosin Regulatory Light Chains; Onset of illness; Paper; Peer Review; Performance; Pharmaceutical Preparations; Phenotype; Phosphorylation; Physiological; Physiology; Play; Preclinical Drug Evaluation; Preparation; Protein Dynamics; Protein Isoforms; Proteins; Publications; Publishing; Recombinants; Regulation; Reporter; Research; Research Personnel; Research Project Grants; Resolution; Role; Skeletal Muscle; Skin; Solutions; Spectrum Analysis; Spin Labels; Stress; Structural Protein; Structure; Techniques; Therapeutic; Time; Training; Transgenic Mice; Vision; Work; X ray diffraction analysis; X-Ray Diffraction; base; career; career development; data acquisition; experience; flexibility; improved; insight; mouse model; myosin-binding protein C; novel; pre-doctoral; programs; protein complex; research study; skeletal; small molecule; translational medicine

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.

描述（由申请人提供）：这是一个名为“心脏肌球蛋白结合蛋白-C的结构动力学”项目的重新提交。该项目的主要目的是为我做好肌肉分子生物物理学独立研究者的职业做好准备，特别强调心脏。我使用肌肉纤维力学和X射线衍射方法的心脏和骨骼肌肉生理的强烈作用，通过光谱探针技术的训练增强了，这是导师实验室的特殊优势。现在，指导计划着重于一个研究项目，该项目综合了我以前的经验，并提出了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）在心脏功能的调节和肥厚性心肌病（HCM）和心力衰竭中的收缩功能缺陷中起主要作用。我的项目还专注于理解和减轻HCM疾病机制，因为有必要解决有关该蛋白质复合物与肌球蛋白和肌动蛋白的结构和动态的基本问题，以及在CPRO相关的HCM疾病发作和进展中随之而来的收缩功能障碍。 该重新提交的三个特定目的是：（1）使用特异性与肌球蛋白或肌动蛋白结合的自旋标签的电子顺磁共振（EPR），以准确测量CPRO遗传消融对皮肤心肌纤维中结构动力学的影响，从几种人HCM的小鼠模型中。 （2）使用目标1的方法来定量确定CPRO对肌球蛋白和肌动蛋白结构动力学的磷酸化的影响。评估在交换的纤维和完整心脏中的基线和磷酸化的收缩性能。 （3）使用偶极电子 - 电子共振（DEER），并将此方法扩展到用肌球蛋白或肌动蛋白溶液中的探针将其扩展到FRET，以定量地确定结合，磷酸化和HCM突变对CPRO特定区域结构动力学的影响。我将使用这些机械细节为CPRO的小分子效应子作为HCM和心力衰竭的潜在疗法提供高通量药物测定的基础。 我最近的PNA出版物揭示了有关CPRO及其磷酸化对标记肌动蛋白丝的结构动态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。所有三个目标都建立了可行性，下面的提案描述了在持续的监督研究经验下实现目标所需的工作 - 在两年内发表几篇高影响力同行评审的论文，增强我在这些研究领域的专业知识，并准备启动我自己的独立实验室。