Site-Specific Mutagenesis of Rat Oncomodulin

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Oligonucleotide-directed mutagenesis will be used to study the structure and properties of the metal binding sites in two, small, calcium-binding proteins that differ in their physiological functions. The protein, oncomodulin, exhibits striking homology to the muscle protein, parvalbumin. Parvalbumin in muscle may be involved in buffering intracellular calcium, but is devoid of the ability to trigger calcium- dependent interactions of contractile proteins. Oncomodulin, on the other hand, is capable of activating cyclic nucleotide phosphodiesterase in a manner reminiscent of calmodulin. This functional difference between the two proteins is mirrored in their metal-binding properties. Oncomodulin possesses one Ca/Mg binding site and one Ca-specific site, while parvalbumin contains two Ca/Mg sites. The objectives of this project are: 1. To determine what factors differentiate a Ca-specific site (a "triggering" site) from a Ca/Mg site (a "relaxing" site), and 2. to identify specific amino acid residues in oncomodulin that are essential to the protein's regulatory capability. An oncomodulin expression plasmid has been constructed, and now single mutations will be introduced into the oncomodulin coding sequence. The effects of these mutations on the protein's metal-binding properties and regulatory ability will then be investigated. Luminescent lanthanide ion probes, together with conventional metal-binding assays, will be used to study the metal-binding properties of the altered proteins. The ability of oncomodulin to stimulate heart cyclic nucleotide phosphodiesterase will be used as a probe of regulatory capability. The family of intracellular calcium-binding proteins includes troponin C, calmodulin, parvalbumin, and vitamin D- dependent calcium-binding protein, in addition to oncomodulin. These proteins display a high degree of homology in their metal- binding domains and are believed to have evolved from a common precursor. There is, however, considerable variation in their metal binding properties. Certain binding sites are quite specific for calcium and do not bind magnesium ion under physiological conditions, while other sites have affinity for both calcium and magnesium. The metal-binding specificity is correlated with physiological function. Calcium specific sites are involved in initiating ("triggering") biological processes, while Ca/Mg sites are not. The molecular basis for the difference in function is not understood. The proposed comparison of the detailed structure of oncomodulin, which has "triggering" activity, and parvalbumin, which does not, should provide important insight into this interesting and physiologically important question.