Structural Dynamics of Muscle Calcium ATPase Regulation

描述（由申请人提供）：本项目的目的是阐明骨骼肌和心脏肌浆网（SR）中活性钙转运的催化和调节的分子机制。重点是Ca-ATP酶（SERCA），将钙泵入SR并因此放松肌肉的大型整体膜酶，以及受磷蛋白（PLB），调节心脏中SERCA的小型整体膜蛋白。该项目的前期工作表明，SERCA活性非常依赖于蛋白质动力学和相互作用。该项目现在测试特定的机制假设，最近获得的X射线和NMR结构。现在的工作越来越多地集中在心脏SR上，因为（a）它是生理学上固有的重要系统，（B）它具有涉及动态蛋白质-蛋白质相互作用的复杂调节机制，以及（c）小蛋白PL B为我们提供了一个很好的机会，将分子遗传学、肽合成和生物物理光谱学的使用联合收割机结合起来。我们将集中在定点标记方法，使用半胱氨酸诱变，荧光融合蛋白，和肽合成。我们将应用互补的光谱方法，包括荧光，磷光，EPR和NMR，分析蛋白质动力学和相互作用。测量将应用于细胞培养中的活细胞膜以及重组膜中的纯化蛋白质。SERCA结构分析的最新进展使我们能够将我们的定点标记实验集中在Ca-ATP酶机制的特定分子模型的测试和修订上。PLB的光谱探针将用于测试和改进其结构，动力学和寡聚体组装的特定模型，受磷酸化和SERCA相互作用的影响。最后，SERCA和PLB上的光谱探针将用于测试和改进监管复合物的结构和动力学的特定模型。拟议的研究汇集了从生物物理学到化学合成再到分子遗传学的强大技术组合，以解决肌肉中钙转运和调节的分子机制。特别是，这项工作对于理解肌肉功能和功能障碍具有根本的重要性。最近的发现表明PLB-SERCA相互作用在心脏病中起着重要作用，我们的研究旨在直接了解潜在治疗方法的分子基础。更一般地说，这个定义明确的系统作为一个模型，用于研究肌肉ATP酶机制和调节中的分子动力学和相互作用的作用，我们正在开发的方法应该证明有效的分析在这一领域的广泛的问题。公共卫生相关性：该项目探讨了肌肉中钙转运调节的基本分子要求，特别关注心脏。具体来说，这项工作的先前见解正被其他人直接用于设计心力衰竭的治疗方法。更一般地说，该项目开发的技术正在应用于涉及肌肉ATP酶系统的广泛的生物医学问题。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the molecular mechanisms of catalysis and regulation of active Ca transport in skeletal and cardiac sarcoplasmic reticulum (SR). The focus is on the Ca-ATPase (SERCA), the large integral membrane enzyme that pumps calcium into the SR and thus relaxes the muscle, and on phospholamban (PLB), the small integral membrane protein that regulates SERCA in the heart. Previous work on this project indicated that SERCA activity is quite dependent on protein dynamics and interactions. This project now tests specific mechanistic hypotheses, informed by recently obtained x-ray and NMR structures. The work is now focused increasingly on cardiac SR, because (a) it is an intrinsically important system physiologically, (b) it features complex regulatory mechanisms involving dynamic protein-protein interactions, and (c) the small protein PLB provides us with an excellent opportunity to combine the use of molecular genetics, peptide synthesis, and biophysical spectroscopy. We will focus on site-directed labeling methods, using Cys mutagenesis, fluorescent fusion proteins, and peptide synthesis. We will apply complementary spectroscopic methods, including fluorescence, phosphorescence, EPR, and NMR, to analyze protein dynamics and interactions. Measurements will be applied to living cell membranes in cell culture as well as to purified proteins in reconstituted membranes. Recent advances in structural analysis of SERCA allow us to focus our site-directed labeling experiments on the testing and revision of specific molecular models for the Ca-ATPase mechanism. Spectroscopic probes of PLB will be used to test and refine specific models for its structure, dynamics, and oligomeric assembly, as affected by phosphorylation and SERCA interaction. Finally, spectroscopic probes on both SERCA and PLB will be used to test and refine specific models for the structure and dynamics of the regulatory complex. The proposed research brings together a powerful combination of techniques, from biophysics to chemical synthesis to molecular genetics, to solve the molecular mechanisms of calcium transport and regulation in muscle. In particular, this work is of fundamental importance for understanding muscle function and malfunction. Recent discoveries indicate that PLB-SERCA interactions play an important role in heart disease, and our research is designed to provide direct insight into the molecular basis of potential therapeutic approaches. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in muscle ATPase mechanism and regulation, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field. PUBLIC HEALTH RELEVANCE: This project explores the fundamental molecular requirements for calcium transport regulation in muscle, with particular focus on the heart. Specifically, previous insights from this work are being used directly by others to design therapeutic approaches for heart failure. More generally, technology developed in this project is being applied to a wide range of biomedical problems involving muscle ATPase systems.