The long terms goals of this grant are to understand the structure and functions of the regulatory (R) subunits of cAMP-dependent protein kinase. To achieve this will require a combination of strategies that extends from mutational analysis, characterization of domains, fluorescence techniques, and high resolution structure analysis. A major advance was made this past granting period when the crystal structure of a deletion mutant (delta 1-91)RI was solved. This structure provides a molecular framework for all of the previous biochemical studies. However, in addition to our increased knowledge about the structure, we have also undergone a major transition in our appreciation of the multiple functions that are contained within this multidomain protein. In addition to being the major receptor for cAMP in eukaryotic cells and the major inhibitor of the catalytic subunit, the R-subunits also help to determine where the kinase will be located within the cell. The region important for subcellular localization is the stable dimerization/docking domain at the amino-terminus which is well removed from the inhibitor site and cAMP-binding domains. Our specific goals over the next granting period are as follows. (1) We shall continue our characterization of the dimerization/docking domain of RI alpha using scanning mutagenesis to determine which residues contribute to the dimer interface and which are critical for binding to anchoring proteins. In conjunction with this systematic probing of functional sites, we shall solve the structure of this domain using NMR. Heterodimers and chimeras will also be used. (2) A second goal will be to rigorously define the docking surfaces that interact with the catalytic subunit. The specific structural differences between RI and RII will also be clarified. (3) The conformational flexibility of the R subunit in the presence and absence of C will be probed. Selected cysteines will be modified with fluorescent probes and used to map binding surfaces. Time resolved fluorescence will be used to define local and global motions. Fluorescence energy transfer will be used to follow interactions between proteins and domains. (4) One of our highest priorities during the next granting period will be to obtain high resolution crystal structures of the full length RI alpha-subunit and of a holoenzyme complex of C and (delta 1-91)RI.
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