The overall goal of the proposed research is to determine the structural basis of Ca-ATPase (calcium pump) regulation mechanisms in both skeletal and cardiac sarcoplasmic reticulum (SR). Calcium is the carrier of contractile stimuli in mammalian organisms. Its homeostatic balance is maintained by Ca-ATPase, a 110 kDa integral membrane protein. There are two small integral membrane proteins that regulate this pump: Phospholamban, a 52-amino-acid phosphoprotein expressed in cardiac muscle, and sarcolipin, a 31-amino-acid protein expressed in skeletal muscle. The goals of this research are to determine the structures of these two proteins in detergent and lipid environments, to determine their interactions with the Ca-ATPase, and to evaluate the structural effects of functionally important modifications, such as mutation and phosphorylation. A multidisciplinary approach will be used, involving solid-phase peptide synthesis, cell culture, site-directed mutagenesis, protein expression and purification, membrane reconstitution, enzyme kinetics, and a combination of solution and solid-state NMR techniques. The following specific aims will be pursued:
AIM 1 : NMR structural determination of phosphorylated and unphosphorylated forms of phospholamban in detergent micelles.
AIM 2 : NMR structural determination of sarcolipin and its variants in detergent micelles.
AIM 3 : Evaluation of the interactions of both phospholamban and sarcolipin with Ca-ATPase in detergent micelles.
AIM 4 : Solid-state NMR determination of the orientations of both phospholamban and sarcolipin in lipid bilayers and their interactions with the Ca-ATPase.The outcomes of this research will be complemented by the structural and dynamics information from site-directed spin and fluorescence labeling studies obtained by our collaborator, Dr. Thomas.Taken with the recently determined high-resolution structure of the Ca-ATPase pump (SERCA1 a) (Toyoshima et al., 2000), the structures of sarcolipin and phospholamban and their interactions with the Ca-ATPase will provide the molecular framework needed to understand calcium transport regulation and its role in muscle function and malfunction. On a more general level, we hope that the approach being developed here - to use a combination of biological and biophysical techniques to probe the functional and structural dynamics of protein-protein interactions within the membrane plane - will have wide application to other important problems in membrane biophysics and physiology.
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