The long term goals are to understand the structure and function of the catalytic (C) subunit of cAMP-dependent protein kinase. By probing this simple protein kinase the investigator also hopes to elucidate many of the general rules for this large family of related enzymes that play critical roles in signal transduction. A major goal for many years was to solve the crystal structure of the C-subunit. Having succeeded during the last granting period represents a major advance. The investigator can now be much more sophisticated in the questions one asks and in the methods one uses to probe the structure. Overall, the objectives are: (1) to understand the role that is played by the highly conserved residues that cluster around the active site and also to determine how these various conserved residues network with one another; (2) to probe the requirements for peptide and protein recognition realizing that the multiple recognition sites are interconnected and that inhibitors will use both common and unique surfaces to achieve high affinity binding; (3) to determine how metals and nucleotides both contribute to the stability of the free C-subunit and to the stabilization of inhibitor complexes; (4) to develop fluorescent probes for monitoring conformational changes. The C-subunit is also regulated by posttranslational modifications, both myristylation and phosphorylation. How these modifications influence both structure and function is a long term objective. Once again, C also is serving as a prototype for understanding in molecular terms how posttranslational modifications influence protein function. Diverse approaches are required to rigorously probe these various sites and domains. Kinetic and biophysical tools will be coupled with mutational analysis to characterize function as well as protein interactions. Specific biophysical tools include analytical gel filtration, Surface Plasmon Resonance, and electrospray mass spectrometry. Developing expression protocols has also been essential, and systems for coexpression and for the expression of fusion proteins of R, PKI, and C are all now available. Finally, the investigator will continue to utilize Xray crystallography and eventually, NMR as definitive tools for understanding the structural consequences of any mutation. In this laboratory the investigators are now in a position to go routinely from the initial mutational analysis to a high resolution crystal structure.
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