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。该项目的主要目标是让我为肌肉分子生物物理学（特别是心脏）的独立研究者的职业生涯做好准备。我在心脏和骨骼肌生理学方面拥有深厚的博士前背景，使用肌纤维力学和 X 射线衍射方法，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殊优势。指导计划现在重点关注一个研究项目，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) 在调节心脏功能以及肥厚型心肌病 (HCM) 和心力衰竭的收缩功能缺陷中发挥着重要作用。我的项目还侧重于了解和缓解 HCM 疾病机制，因为有必要解决有关该蛋白质与肌球蛋白和肌动蛋白复合物的结构和动力学的基本问题，以及 CPro 相关 HCM 疾病发作和随后出现的收缩功能障碍。进展。 本次重新提交有三个具体目标：(1) 使用与肌球蛋白或肌动蛋白特异性结合的自旋标签的电子顺磁共振 (EPR)，准确测量 CPro 基因消融对带皮心肌纤维结构动力学的影响，从多种角度人类 HCM 小鼠模型。 (2) 使用目标 1 的方法定量确定 CPro 的 PKA 依赖性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) 并将该方法扩展到肌球蛋白或肌动蛋白溶液中 CPro 探针的 FRET，定量确定结合、磷酸化和 HCM 突变对 CPro 特定区域结构动力学的影响。我将利用这些机制细节为 CPro 小分子效应物的高通量药物测定提供基础，作为 HCM 和心力衰竭的潜在疗法。 我最近在 PNAS 上发表的文章揭示了有关 CPro 及其磷酸化对标记肌动蛋白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。所有三个目标都已确立可行性，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，并准备创办自己的独立实验室。