Biophysical Studies of Membrane Molecular Dynamics

描述(申请人提供)：本研究的目标是确定骨骼肌和心肌中肌浆网(SR)活性钙转运的催化和调节的分子机制。关注的焦点是Ca-ATPase(SERCA)，这是一种将钙泵入SR从而放松肌肉的大的整膜酶，以及磷蛋白(PLB)和肌磷脂(SLN)，这是调节SERCA的小的整膜蛋白。我们将特别关注心脏，在那里PLB和SLN都被认为扮演着影响心脏健康的重要调节角色。我们将测试特定的机械假设，了解结构动力学在这个系统中的功能作用。我们将重点介绍定点标记方法，使用细胞培养、诱变、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。我们将追求以下具体目标：(1)发展改进的光谱方法来研究膜分子动力学，以肌浆网(SR)为模型系统来演示这些技术。(2)探索SERCA的结构动力学，以确定分子运动和相互作用的变化是如何耦合到Ca-ATPase反应周期的。(3)探讨PLB与SERCA和SLN的功能相互作用对其结构动力学的影响。(4)探讨SERCA与PLB和SLN的功能相互作用对SERCA结构的影响。AIMS 3和AIMS 4将使我们能够测试钙泵调节机制的特定模型。 这项拟议的研究汇集了从生物物理学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，特别是与心脏病相关的问题具有重要意义。更广泛地说，这个定义明确的系统作为研究分子动力学和相互作用在膜蛋白机制和调控中的作用的模型，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。

DESCRIPTION (provided by applicant): The goal of this research is to determine the molecular mechanisms of catalysis and regulation of active calcium transport in sarcoplasmic reticulum (SR) in skeletal and cardiac muscle. 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 phospholamban (PLB) and sarcolipin (SLN), the small integral membrane proteins that regulate SERCA. We will focus particular attention on the heart, where both PLB and SLN are proposed to play important regulatory roles that affect cardiac health. We will test specific mechanistic hypotheses for the functional roles of structural dynamics in this system. We will focus on site-directed labeling methods, using cell culture, 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. We will pursue the following specific aims: (1) Develop improved spectroscopic methods for studying membrane molecular dynamics, using sarcoplasmic reticulum (SR) as a model system to demonstrate these techniques. (2) Probe the structural dynamics of SERCA, to determine how changes in molecular motions and interactions are coupled to the Ca-ATPase reaction cycle. (3) Probe the structural dynamics of PLB, as affected by functional interactions with SERCA and SLN. (4) Probe the structural dynamics of SERCA, as affected by functional interactions with PLB and SLN. Aims 3 and 4 together will allow us to test specific models for the mechanisms of calcium pump regulation. The proposed research brings together a powerful combination of techniques, from biophysics 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, with particular relevance to heart disease. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in membrane protein mechanism and regulation, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.