The goal of this project is to elucidate the molecular mechanisms of catalysis and regulation of active Ca transport in skeletal and cardiac sarcoplasmic reticulum (SR). 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 on phospholamban (PLB), the small integral membrane protein that regulates SERCA in the heart. Previous work on this project indicated that SERCA activity is quite dependent on protein dynamics and interactions. This project now tests specific mechanistic hypotheses, informed by recently obtained x-ray and NMR structures. The work is now focused increasingly on cardiac SR, because (a) it is an intrinsically important system physiologically, (b) it features complex regulatory mechanisms involving dynamic protein-protein interactions, and (c) the small protein PLB provides us with an excellent opportunity to combine the use of molecular genetics, peptide synthesis, and biophysical spectroscopy. We will focus on site-directed labeling methods, using Cys 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. Measurements will be applied to living cell membranes in cell culture as well as to purified proteins in reconstituted membranes. Recent advances in structural analysis of SERCA allow us to focus our site-directed labeling experiments on the testing and revision of specific molecular models for the Ca-ATPase mechanism. Spectroscopic probes of PLB will be used to test and refine specific models for its structure, dynamics, and oligomeric assembly, as affected by phosphorylation and SERCA interaction. Finally, spectroscopic probes on both SERCA and PLB will be used to test and refine specific models for the structure and dynamics of the regulatory complex. The proposed research brings together a powerful combination of techniques, from biophysics to chemical synthesis 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. Recent discoveries indicate that PLB-SERCA interactions play an important role in heart disease, and our research is designed to provide direct insight into the molecular basis of potential therapeutic approaches. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in muscle ATPase mechanism and regulation, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.
This project explores the fundamental molecular requirements for calcium transport regulation in muscle, with particular focus on the heart. Specifically, previous insights from this work are being used directly by others to design therapeutic approaches for heart failure. More generally, technology developed in this project is being applied to a wide range of biomedical problems involving muscle ATPase systems.
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