Structural Dynamics of Muscle Calcium ATPase Regulation

肌肉钙 ATP 酶调节的结构动力学

• 批准号: 8598763
• 负责人: David D Thomas
• 金额: $ 64.12万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598763
• 关键词: ATP phosphohydrolase; Active Biological Transport; Affect; Affinity; Attention; Back; Binding; Biochemical; Biological Assay; Biology; Biophysics; Ca(2+)-Transporting ATPase; Calcium; Cardiovascular Diseases; Catalysis; Cells; Chemicals; Chimeric Proteins; Clinical Trials; Complement; Complex; Computer Simulation; Data; Deer; Development; Diabetes Mellitus; Electron Spin Resonance Spectroscopy; Engineering; Enzymes; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Future; Goals; Health; Heart; Heart failure; Homeostasis; Integral Membrane Protein; Label; Life; Ligands; Light; Link; Malignant Neoplasms; Measurement; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Genetics; Motion; Motivation; Muscle; Muscle function; Muscular Dystrophies; Mutagenesis; Mutation; Myopathy; Peptide Synthesis; Phosphoproteins; Phosphorylation; Play; Proteins; Pump; Reaction; Reader; Records; Regulation; Research; Resolution; Roentgen Rays; Role; Sarcoplasmic Reticulum; Scanning; Scientist; Site; Spectrum Analysis; Striated Muscles; Structural Models; Structural Protein; Structure; Study models; System; Techniques; Technology; Testing; Therapeutic; Time; Translational Research; Work; base; calcium disorder; design; drug development; drug discovery; feeding; gene therapy; high throughput screening; innovation; insight; molecular dynamics; mutant; new technology; novel; phospholamban; phosphorescence; pre-clinical; protein protein interaction; protein structure; public health relevance; reconstitution; response; simulation; small molecule; therapeutic target; therapy design; tool

DESCRIPTION (provided by applicant): This project's goal is to determine the protein structures, dynamics, and interactions that govern the active transport of calcium in muscle, to pave the way for rational therapeutic design in muscle disease. We focus on two integral membrane proteins, the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) and its principal regulator in the heart, phospholamban (PLB). Our core technology is site-directed spectroscopy in both purified proteins and living cells. We will develop and apply innovative and complementary methods in site-directed labeling, fluorescence, and EPR, combined with NMR and crystallographic data obtained from our collaborators. All of this will be integrated and informed by computational simulations of both spectroscopic and molecular dynamics to analyze protein structure, dynamics, and interactions. Aims 1 and 2 deal with applications to fundamental mechanisms in this system, and Aims 3 and 4 combine these techniques and fundamental insights to move toward biophysically designed therapies. In Aim1, we investigate the fundamental functional dynamics of SERCA, to determine the key transitions in structure and motion that govern its catalytic mechanism, {focusing on steps that are crucial for substrate activation and regulation}. Aim2 investigates the mechanism by which PLB regulates SERCA, adding themes of protein-protein interaction and phosphorylation. Aim3 uses the insights of the first two aims, based primarily on discoveries of the previous project period, to design PLB mutants that are strong candidates for future gene therapy applications. Similarly, Aim4 uses live-cell fluorescence assays of SERCA and PLB, in conjunction with our new technology in high-throughput fluorescence lifetime measurement, to develop and apply novel small- molecule screens, with the ultimate goal of drug discovery for treatment of heart failure and other disorders of calcium homeostasis. {The four Aims are synergistic, strengthening each other with new discoveries and hypotheses to be tested, but they are not interdependent, since feasibility has been established independently for each aim and subaim.} This research brings together a powerful combination of techniques, concepts, and collaborators, from biophysics to chemical biology to molecular genetics, to solve the molecular mechanisms of calcium transport and regulation in muscle. This project remains grounded in fundamental biophysical mechanisms, but it has recently become clear that SERCA is one of the most important therapeutic targets for some of the greatest health problems, including heart failure, muscular dystrophy, diabetes, and cancer. It is also clear that the biophysical tools being developed in this project have matured to the point where they can play a crucial role not only in understanding the functions of SERCA and PLB, but also in controlling these functions. Therefore, the collaborative team now includes scientists with strong track records in drug development and gene therapy. It is anticipated that by the end of the next funding period, this project will stimulate separate efforts in truly translational research on muscle therapeutics.

描述（由申请人提供）：该项目的目标是确定蛋白质结构，动力学和相互作用，这些蛋白质结构，动力学和相互作用控制肌肉中钙的主动转运，为肌肉疾病的合理治疗设计铺平道路。我们专注于两个不可或缺的膜蛋白，肌浆网（SR）钙ATP酶（SERCA）和它的主要调节器在心脏，受磷蛋白（PLB）。我们的核心技术是在纯化蛋白质和活细胞中进行定点光谱分析。我们将开发和应用创新和互补的方法，在定点标记，荧光和EPR，结合NMR和晶体学数据从我们的合作者。所有这些都将通过光谱和分子动力学的计算模拟来整合和通知，以分析蛋白质结构，动力学和相互作用。目标1和2涉及该系统中基本机制的应用，目标3和4联合收割机将这些技术和基本见解结合起来，朝着生物医学设计的治疗方向发展。在Aim 1中，我们研究了SERCA的基本功能动力学，以确定控制其催化机制的结构和运动的关键转变，{专注于对底物活化和调节至关重要的步骤}。Aim 2研究PLB调节SERCA的机制，增加了蛋白质-蛋白质相互作用和磷酸化的主题。Aim 3利用前两个目标的见解，主要基于前一个项目期间的发现，设计PLB突变体，这些突变体是未来基因治疗应用的强有力候选者。同样，Aim 4使用SERCA和PLB的活细胞荧光测定，结合我们在高通量荧光寿命测量中的新技术，开发和应用新型小分子筛选，最终目标是发现治疗心力衰竭和其他钙稳态紊乱的药物。{The四个目标是协同的，通过新的发现和有待检验的假设相互加强，但它们并不相互依赖，因为每个目标和子目标的可行性都是独立建立的。这项研究汇集了技术，概念和合作者的强大组合，从生物物理学到化学生物学再到分子遗传学，以解决肌肉中钙转运和调节的分子机制。该项目仍然立足于基本的生物物理机制，但最近已经清楚，SERCA是一些最大的健康问题，包括心力衰竭，肌肉萎缩症，糖尿病和癌症的最重要的治疗目标之一。同样清楚的是，该项目正在开发的生物物理工具已经成熟， 在这一点上，它们不仅在理解SERCA和PLB的功能方面，而且在控制这些功能方面发挥着至关重要的作用。因此，合作团队现在包括在药物开发和基因治疗方面具有良好记录的科学家。预计到下一个资助期结束时，该项目将刺激肌肉治疗学真正转化研究的单独努力。