OPTICAL STUDIES OF PROTEIN STRUCTURE/FUNCTION

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The structure and mechanism of action of a family of myristoylated calcium-sensing proteins, exemplified by retinal recoverin, will be investigated by fluorescence, NMR, and spin-label spectroscopy. These acylated proteins move from the cytosol to a membrane on binding calcium. The aim is to determine the molecular mechanism of calcium-myristoyl switches and define their roles in signal transduction processes. Having solved the three-dimensional structures of calcium-free myristoylated recoverin and calcium-bound unmyristoylated recoverin, we turn now to the determination of the structure of the calcium-bound form of myristoylated recoverin to reveal the precise structural basis of the switch. Our working hypothesis is that recoverin and brain homologs such as neurocalcin and hippocalcin serve to couple calcium cascades to G-protein cascades by interacting with kinases that deactivate seven-helix receptors. The membrane-contact sites of recoverin in the Ca2+-bound state and their depth will be determined by spin-label ESR spectroscopy using nitroxide-labeled myristate and nitroxide-tagged cysteines introduced by mutagenesis. Structural, spectroscopic, and functional studies of the interaction of Ca2+-bound recoverin with rhodopsin kinase and fragments of this target will also be carried out. Three classes of recoverin mutants will be generated and analyzed to further our understanding of the structure and dynamics of this sensor: (1) Nonpolar residues in the myristoyl binding pocket will be replaced by polar ones. (2) Glycines in putative hinge regions will be changed to alanine. (3) Charged residues surrounding a concave hydrophobic surface, the putative target-binding site, will be charged to oppositely-charged residues. Structural studies of neurocalcin and hippocalcin will also be carried out and targets of these brain homologs will be identified using the yeast two-hybrid system. A yeast homolog will be expressed and analyzed to provide insight into the evolution of this family of neuronal calcium sensors.