The overall goal of this research program is to develop novel fluorescence techniques and to use them in concert with other experimental approaches to elucidate the structure and dynamics of calcium-sensing proteins. Recoverin, a recently discovered member of the EF hand superfamily of calcium-binding proteins, plays a key role in vision by activating guanylate cyclase when the cytosolic calcium level is lowered following illumination. The presence of antibodies to recoverin in patients with cancer-associated retinopathy, an autoimmune retinal degenerative disease induced by peripheral tumors, suggests that homologs of recoverin are widely distributed. We have recently cloned recoverin cDNA and obtained high level expression of functional protein in E. coli. The following biophysical, biochemical, and molecular genetic studies will be carried out: (1) The kinetics, thermodynamics, and cooperativity of calcium binding will be determined fluorimetrically and microcalorimetrically. (2) Calcium-induced conformational transitions will be detected by picosecond emission anisotropy measurements, fluorescence energy transfer, and low- angle x-ray scattering. (3) Large quantities of recombinant recoverin will be produced to obtain crystals for x-ray analysis. (4) Site-specific mutants will be generated and analyzed to identify residues that are critical for calcium binding, conformational switching, and target binding. (5) The folding of recoverin will be investigated by fluorescence spectroscopy and other physical methods to establish the temporal sequence of compaction and acquisition of secondary and tertiary structure. (6) cDNA libraries and expression libraries will be screened for homologs of recoverin outside the retina. Another major aim of this research is to use fluorescence spectroscopy to elucidate how type II multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is activated by Ca2+- calmodulin and by autophosphorylation. CaM kinase, a major brain protein, regulates neurotransmitter synthesis and release, and also plays key roles in peripheral tissues, where it controls membrane channels. The proposed research will give insight into the mechanism of transduction of calcium signals and provide a basis for designing therapeutic agents that selectively modify particular pathways. We will also continue to develop new methods for high-sensitivity fluorescence detection. Picosecond laser excitation and microchannel plate detection will be used to enhance rejection of background scattering. Fluorescence detection of single molecules such as phycoerythrin-labeled antibodies and DNA probes will open new vistas in medical diagnostics.
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