The primary objective of this proposal is to utilize directed mutagenesis as a tool for understanding the multiple functional sites associated with the regulatory subunit of cAMP-dependent protein kinase I. A full length cDNA clone for the regulatory subunit is now being inserted into various expression vectors - initially into pUC8 and then into the tac promotor. Both systems will be tested for expression of full length, functional protein. Expressing clones will be identified immunologically using Western Blots. Once expressing clones have been identified, RI will be purified from these clones and the functional properties of RI characterized by affinity labeling, limited proteolysis, reassociation with catalytic subunit, and binding of cAMP analogs. Having isolated a suitable expression system, we shall procede to use mutagenesis techniques to produce variants of RI. Specific functional sites to be probed include 1) two in tandem cAMP binding sites, 2) the R-C interaction site, and 3) the R-R interaction site. Methods for mutagenesis will include the site-directed mutation of single amino acid residues. Mutations will be made by synthesizing oligonucleotide probes that code for single amino acid changes in the cAMP binding site. The crystal structure of the bacterial cAMP binding protein, CAP, which on the basis of amino acid sequence is clearly homologous to the R-subunit, will provide an initial framework for mutagenesis in the two cAMP binding domains. The objective here will be to identify specific residues that participate in cAMP binding and more importantly to ascertain the importance or relevance of the two cAMP binding sites on each protomer. A second mutagenesis approach will be to synthesize a mixed oligonucleotide probe for a defined region, thereby isolating a number of variants each containing a single amino acid change in a single experiment. Both of these approaches will be used to mutate residues in other functional sites in the protein. An additional group of deletion variants will be constructed that lack various regions of the protein. One such truncated protein will lack the second cAMP-binding domain. An independent cAMP binding domain will also be isolated as a model protein for cAMP-binding. Finally, a fragment will be isolated that retains the R-C interaction site but that lacks both cAMP binding domains, and its ability to inhibit the C-subunit will be characterized. A long-range objective will be to crystallize the RI subunit and to initiate x-ray crystallographic studies.
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