Structural Dynamics of Cardiac Calcium Transport Regulation

描述（由申请人提供）：本研究的目的是确定心脏肌浆网（SR）中活性钙转运调节的分子机制，具体目标是生物化学、分子生物学和生物物理学博士论文。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SERCA的小型整体膜蛋白。具体的机制假设将被测试在这个系统中的结构动力学的功能作用。该方法是将顺磁探针（氮氧自旋标记）连接到PLB的合成变体上的选定位点，在存在或不存在SERCA的情况下将自旋标记的PLB重组成脂质双层，并进行电子顺磁共振（EPR）实验以探测PLB的结构和动力学。我将重点介绍PLB胞质结构域的几个方面，这些结构域最近被提出在心脏功能中发挥重要作用：（a）使用合成掺入到PL B骨架中的自旋标记，将检测胞质结构域的螺旋区段中的动态有序-无序转变。（B）通过检测自旋标记对之间的偶极相互作用，我将测量自旋-自旋距离，从而检测寡聚体结构和肽链延伸。（c）通过测量膜结合的顺磁弛豫剂对自旋标记饱和度的影响，我将探测胞质结构域和膜表面之间的动态相互作用。这三种生物物理分析将与功能测量相结合，以实现以下三个目标：（1）比较PLB单体和五聚体的结构动力学，以解决目前关于五聚体结构的争议。(2)PLB与SERCA相互作用时的功能动力学测试模型。(3)在存在和不存在SERCA的情况下PLB磷酸化对其结构动力学影响的测试模型。 这项研究的重点是两种心脏蛋白质，它们在各种心脏疾病中起着核心作用。特别是，目前的医学研究已经确定受磷蛋白作为心力衰竭治疗方法的主要靶标。这项研究的结果将提供合理设计药物、基因疗法和其他治疗衰竭心脏的方法所需的详细分子信息。

DESCRIPTION (provided by applicant): The goal of this research is to determine the molecular mechanism of active calcium transport regulation in cardiac sarcoplasmic reticulum (SR), with the specific goal of a PhD thesis in Biochemistry, Molecular Biology, and Biophysics. 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), the small integral membrane protein that regulates SERCA. Specific mechanistic hypotheses will be tested for the functional roles of structural dynamics in this system. The approach is to attach paramagnetic probes (nitroxide spin labels) to selected sites on synthetic variants of PLB, reconstitute spin-labeled PLB into lipid bilayers in the presence or absence of SERCA, and perform electron paramagnetic resonance (EPR) experiments to probe the structure and dynamics of PLB. I will focus on several aspects of PLB cytoplasmic domain structure that have recently been proposed to play important roles in cardiac function: (a) Using a spin label synthetically incorporated into the PLB backbone, I will detect dynamic order-disorder transitions in the helical segments of the cytoplasmic domain, (b) By detecting the dipolar interaction between pairs of spin labels, I will measure spin-spin distances and thus detect oligomeric structures and peptide chain extension, (c) By measuring the effect of membrane-bound paramagnetic relaxation agents on spin label saturation, I will probe the dynamic interaction between the cytoplasmic domain and the membrane surface. These three biophysical assays will be combined with functional measurements to pursue the following three aims: (1) Compare the structural dynamics of PLB monomer and pentamer, to resolve a current controversy about the structure of the pentamer. (2) Test models for functional dynamics of PLB while interacting with SERCA. (3) Test models for the effect of PLB phosphorylation on its structural dynamics in the presence and absence of SERCA. This research focuses on two cardiac proteins that play central roles in a wide range of heart diseases. In particular, current medical research has identified phospholamban as a major target of therapeutic approaches to heart failure. The findings of this research will provide the kind of detailed molecular information that is needed for the rational design of drugs, gene therapies, and other means of treating the failing heart.