The objectives of this proposal are to use the tools of molecular biology to better understand the relationship between structure and function in the regulatory (R) subunit of cAMP-dependent protein kinase. The R-subunit exists as part of an inactive holoenzyme complex with the catalytic (C) subunit. In the presence of cAMP, this holoenzyme (R2C2) dissociates into an R2-dimmer and 2 active C-subunits. The R-subunit has a well defined domain structure. The N-terminal region that contains the site of contact between the 2 protomers in the dimmer is followed by 2 tandem cAMP binding domains. A proteolytically sensitive hinge region, which lies in the NH2-terminal segment, has a site that is essential for interaction with the C-subunit. An expression vector has been constructed which yields up to 40 mg/ml of soluble R-subunit per liter of E. coli, and mutations are being introduced into the R- subunit. Oligonucleotide probes are being used to introduce site- specific changes and deletions. Random mutagenesis methods also will be used. Methods to screen for function such as cAMP binding also are being developed. Specific questions that are being asked include: 1) What residues contribute to the immediate environment of each cAMP binding site? 2) What are the conformational changes that are induced as a consequence of cAMP binding and which lead to the dissociation of C? 3) What are the functional domains and can they be isolated as discrete structural entities which retain function? 4) What are the points of contact between the R- and C- subunits? 5) What are the points of contact between the 2 R- subunits in the dimmer? Specific sites for mutation have been chosen on the basis of extensive mapping of the cAMP binding sites with analogs of cAMP and on the basis of specific sites of covalent modification following photoaffinity labeling with 8-N3-cAMP. Limited proteolysis has provided a preliminary indication of the domain structure. Our interpretation of the cAMP binding domains has been facilitated by the homologies that each domain shares with the catabolite gene activation protein (CAP), and a model has been constructed by building the R-sequences into the crystallographic coordinates of CAP. Mutant R-subunits which show unique phenotypic properties will be overexpressed in cultured cells in an effort to better understand cAMP-mediated functions.
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