Biophysical Studies of Membrane Molecular Dynamics

描述（由申请人提供）：本研究的目的是确定骨骼肌和心肌肌浆网（SR）中活性钙转运的催化和调节的分子机制。重点是Ca-ATP酶（SERCA），将钙泵入SR并因此松弛肌肉的大的完整膜酶，以及受磷蛋白（PLB）和肌磷脂（SLN），调节SERCA的小的完整膜蛋白。我们将特别关注心脏，其中PLB和SLN都被认为是影响心脏健康的重要调节作用。我们将测试特定的机制假设的功能作用的结构动力学在这个系统中。我们将集中在定点标记方法，使用细胞培养，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。我们将致力于以下具体目标：（1）发展用于研究膜分子动力学的改进的光谱方法，使用肌浆网（SR）作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如何与Ca-ATP酶反应循环相耦合。(3)探索PLB的结构动力学，受到与SERCA和SLN的功能相互作用的影响。(4)探索SERCA的结构动力学，受PLB和SLN功能相互作用的影响。目标3和4一起将使我们能够测试钙泵调节机制的特定模型。 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，以解决肌肉中钙转运和调节的分子机制。特别是，这项工作对于了解肌肉功能和功能障碍具有根本重要性，特别是与心脏病有关。更一般地说，这个定义明确的系统作为一个模型，用于研究分子动力学和相互作用的作用，在膜蛋白的机制和调节，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。

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.