Structural Dynamics of Muscle Calcium ATPase Regulation

描述（由申请人提供）：该项目的目标是确定控制肌肉中钙主动运输的蛋白质结构、动力学和相互作用，为肌肉疾病的合理治疗设计铺平道路。我们专注于两种完整的膜蛋白，肌浆网ca - atp酶（SERCA）及其在心脏中的主要调节因子，磷蛋白（PLB）。我们的核心技术是纯化蛋白和活细胞的定点光谱学。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，结合从我们的合作者获得的核磁共振和晶体学数据。所有这些都将通过光谱和分子动力学的计算模拟来分析蛋白质结构、动力学和相互作用。目标1和目标2涉及该系统中基本机制的应用，目标3和目标4将这些技术和基本见解结合起来，走向生物物理设计的治疗方法。在ai1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，重点关注对底物激活和调节至关重要的步骤。目的2研究了PLB调控SERCA的机制，增加了蛋白相互作用和磷酸化的主题。Aim3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有力候选者。同样，Aim4利用SERCA和PLB的活细胞荧光分析，结合我们的高通量荧光寿命测量新技术，开发和应用新的小分子筛选，最终目标是发现治疗心力衰竭和其他钙稳态紊乱的药物。{这四个目标是协同的，通过有待检验的新发现和假设相互加强，但它们不是相互依存的，因为每个目标和子目标的可行性都是独立建立的。这项研究汇集了从生物物理学到化学生物学到分子遗传学的技术、概念和合作者的强大组合，以解决肌肉中钙运输和调节的分子机制。该项目仍以基本的生物物理机制为基础，但最近已经清楚地表明，SERCA是一些重大健康问题（包括心力衰竭、肌肉萎缩症、糖尿病和癌症）最重要的治疗靶点之一。同样清楚的是，在这个项目中开发的生物物理工具已经成熟到可以

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.