Structural Dynamics of Muscle Calcium ATPase Regulation

描述(申请人提供)：本项目的目标是阐明骨骼和心肌肌浆网(SR)中活性钙转运的催化和调节的分子机制。重点是钙-三磷酸腺苷酶(SERCA)，这是一种将钙泵入SR从而放松肌肉的大型完整膜酶，以及磷蛋白(PLB)，这是一种调节心脏中SERCA的小的完整膜蛋白。该项目以前的工作表明，SERCA的活性在很大程度上取决于蛋白质的动力学和相互作用。这个项目现在通过最近获得的X射线和核磁共振结构来测试特定的机械假说。这项工作现在越来越多地集中在心脏SR上，因为(A)它是一个内在重要的生理系统，(B)它具有复杂的调节机制，涉及动态的蛋白质-蛋白质相互作用，以及(C)小蛋白PLB为我们提供了一个极好的机会来结合分子遗传学、肽合成和生物物理光谱学的使用。我们将重点介绍定点标记方法，使用半胱氨酸突变、荧光融合蛋白和多肽合成。我们将应用互补光谱方法，包括荧光、磷光、EPR和核磁共振，来分析蛋白质的动力学和相互作用。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测试和修订特定的Ca-ATPase机制分子模型上。PLB的光谱探针将被用来测试和完善其结构、动力学和低聚物组装的特定模型，这些模型受到磷酸化和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完善调控复合体的结构和动力学的特定模型。这项拟议的研究汇集了从生物物理到化学合成再到分子遗传学的强大技术组合，以解决肌肉中钙运输和调节的分子机制。特别是，这项工作对于了解肌肉功能和功能障碍具有重要意义。最近的发现表明，PLB-SERCA相互作用在心脏病中发挥着重要作用，我们的研究旨在为潜在治疗方法的分子基础提供直接的见解。更广泛地说，这个定义明确的系统作为一个模型，研究分子动力学和相互作用在肌肉ATPase机制和调控中的作用，我们正在开发的方法应该被证明在分析这一领域的广泛问题方面是有效的。与公共健康相关：这个项目探索了肌肉中钙运输调节的基本分子要求，特别是心脏。具体地说，这项工作之前的见解正被其他人直接用于设计心力衰竭的治疗方法。更广泛地说，在这个项目中开发的技术正在被应用于涉及肌肉ATPase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.