Structural Dynamics of Muscle Calcium ATPase Regulation

项目描述（由申请人提供）：本项目旨在阐明骨和心脏肌浆网（SR）中活性钙转运的催化和调控分子机制。重点是钙三磷酸腺苷酶（SERCA），一种将钙泵入SR从而放松肌肉的大型整体膜酶，以及磷蛋白（PLB），一种调节心脏中SERCA的小型整体膜蛋白。该项目的前期工作表明，SERCA活性很大程度上依赖于蛋白质动力学和相互作用。根据最近获得的x射线和核磁共振结构，该项目现在测试特定的机械假设。这项工作现在越来越多地集中在心脏SR上，因为(a)它在生理上是一个内在重要的系统，(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PLB为我们提供了结合分子遗传学，肽合成和生物物理光谱学使用的绝佳机会。我们将重点关注位点定向标记方法，使用Cys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和核磁共振，来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位点定向标记实验集中在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完善其结构、动力学和寡聚物组装的特定模型，这些模型受磷酸化和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完善调节复合物的结构和动力学的特定模型。本研究将从生物物理学到化学合成再到分子遗传学等一系列强有力的技术结合在一起，以解决肌肉中钙转运和调节的分子机制。特别是，这项工作对理解肌肉功能和功能障碍至关重要。最近的发现表明，PLB-SERCA相互作用在心脏病中起着重要作用，我们的研究旨在为潜在治疗方法的分子基础提供直接的见解。更一般地说，这个定义良好的系统可以作为研究分子动力学和相互作用在肌肉atp酶机制和调节中的作用的模型，我们正在开发的方法应该在分析该领域的广泛问题中被证明是有效的。公共卫生相关性：该项目探索肌肉中钙运输调节的基本分子需求，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。更广泛地说，在这个项目中开发的技术正在广泛应用于涉及肌肉atp酶系统的生物医学问题。

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.