Structural Dynamics of Cardiac Calcium Transport Regulation

描述(申请人提供)：这项研究的目标是确定心肌肌浆网(SR)中主动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。关注的焦点是Ca-ATPase(SERCA)和磷蛋白(PLB)，SERCA是将钙泵入SR从而放松肌肉的大型完整膜酶，PLB是调节SERCA的小型完整膜蛋白。具体的力学假设将被检验结构动力学在这个系统中的功能作用。该方法是将顺磁性探针(氮氧化物自旋标记)连接到PLB的合成变体上，在SERCA存在或不存在的情况下，将自旋标记的PLB重新构建为脂质双层，并进行电子顺磁共振(EPR)实验来探测PLB的结构和动力学。我将集中讨论最近被提出在心脏功能中发挥重要作用的PLB细胞质结构域的几个方面：(A)利用被人工整合到PLB主干中的自旋标记，我将检测细胞质结构域螺旋片段中的动态有序-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，从而检测寡聚结构和肽链的延伸；(C)通过测量膜结合顺磁性松驰剂对自旋标记饱和度的影响，我将探索细胞质结构域与膜表面之间的动态相互作用。这三种生物物理方法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SERCA存在和不存在的情况下，PLB磷酸化对其结构动力学影响的测试模型。 这项研究的重点是在广泛的心脏疾病中发挥核心作用的两种心脏蛋白。特别是，目前的医学研究已经确定磷蛋白是心力衰竭治疗方法的主要靶点。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心力衰竭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.