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.
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