Protein phosphorylation is a major mechanism for regulation in eukaryotic cells, and the protein kinases represent a large and diverse family of enzymes that include many oncogenic proteins. cAMP-dependent protein kinase (cAPK) was one of the first protein kinases to be discovered, and it remains as one of the simplest and biochemically best understood. Furthermore, sequence similarities reveal that all eukaryotic protein kinases, despite their diversity, have evolved in part from a common precursor. Since this conserved catalytic core corresponds to most of the catalytic (C) subunit of cAPK, this simple kinases can serve as a framework for the entire family. Our objectives are to better define the functional sites that are associated with the C-subunit using a combination of chemical, biophysical and recombinant techniques. A particular focus in the MgATP binding site will be the glycine-rich segment thought to interact with the phosphates of ATP. Having demonstrated fluorescence changes associated with MgATP binding, we shall compare numerous analogs of ATP for their capacity to induce similar changes. Included in these analogs will be ADP.VO4 which has the potential to selectively cleave the polypeptide chain by a photo-induced mechanism. Mutations will also be introduced into the glycine-rich loop. Asp 184, a good candidate for the base catalyst will also be replaced. Based on reactivity with a water soluble carbodiimide, several regions have been targeted as potential sites for peptide recognition. These will be selectively replaced and the consequences on peptide recognition evaluated. A goal here is to selectively change peptide specificity. In addition to characterizing functional specific sites, we shall attempt to better define the conformational changes that are associated with substrates binding to the C-subunit. To accomplish this we shall use a combination of neutron scattering, small angle X-ray scattering, fluorescence changes and analytic gel chromatography. Finally, we shall define more precisely the interactions between the R- and C-subunits in the type I holoenzyme. The high affinity MgATP binding site will be mapped by a combination of affinity labeling and group-specific labeling as well as with analogs of MgATP. We also shall use subunits labeled with fluorescent probes to directly monitor subunit interactions. Superimposed on the above goals is our intention to continue to facilitate the crystallographic studies by providing wild type and mutant proteins in large quantities.
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