Structural Determinants of Calcium Pump Regulation

描述（由申请人提供）：申请人提出了一种2-AIM策略，以探索SERCA钙泵通过其主要结合伴侣PLB调节的分子机制。AIM 1描述了使用荧光共振能量转移（FRET）来定量PLB结合相互作用的亲和力和所得蛋白质复合物的结构。所提出的实验直接测量PLB的磷酸化和突变对其调节相互作用的影响。由于荧光探针是遗传编码的，因此首次在活细胞中观察到这些膜蛋白相互作用。AIM 2建议测量对心脏Ca 2+调节重要的膜蛋白复合物的蛋白结合动力学。这些膜蛋白复合物的形成和溶解速率是其功能的重要决定因素，但这些速率无法用经典的蛋白质-蛋白质结合动力学方法进行测定。申请人发明了一种新的光学方法，可以测量活细胞中的膜蛋白亚基交换动力学。公共卫生相关性：蛋白质PLB和SERCA具有临床意义，因为它们在心脏功能和疾病中起核心作用。这些蛋白质的紊乱与心力衰竭有关，人类PLB的缺失或突变导致疾病“扩张型心肌病”。因此，PLB被认为是治疗心力衰竭的高价值治疗靶标，心力衰竭是美国的主要死亡原因。

DESCRIPTION (provided by applicant): The applicant proposes a 2-AIM strategy to explore molecular mechanisms of SERCA calcium pump regulation by its major binding partner PLB. AIM1 describes the use of fluorescence resonance energy transfer (FRET) to quantify the AFFINITY of PLB binding interactions and the STRUCTURE of the resulting protein complexes. The proposed experiments directly measure the effect of phosphorylation and mutation of PLB on its regulatory interactions. Because the fluorescent probes are genetically encoded, observations of these membrane protein interactions are being made in living cells for the first time. AIM2 proposes to measure the PROTEIN BINDING KINETICS of the membrane protein complexes important for cardiac Ca2+ regulation. The rate of formation and dissolution of these membrane protein complexes is an important determinant of their function, but these rates are inaccessible to classical protein-protein binding kinetics methods. The applicant has invented a new optical method that can measure membrane protein subunit exchange dynamics in living cells. PUBLIC HEALTH RELEVANCE: The proteins PLB and SERCA are of clinical significance because of their central role in cardiac function and disease. Disorders of these proteins are associated with heart failure, and loss or mutation of PLB in humans results in the disease "dilated cardiomyopathy". Thus, PLB is a regarded as a high value therapeutic target in the treatment of heart failure, a leading cause of death in the United States.