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.
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. ? ? ?
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