This proposal will extend the knowledge of the structure, in solution, of the protein kinases, Csk, and Abelson (Abl), and their complexes in intracellular signal transduction using contemporary NMR and molecular biology approaches, and new technologies. The last will extend segmental labeling using expressed protein ligation, and direct determination of segmental motion of multidomain proteins from relaxation studies and from residual dipolar couplings. The kinases to be studied and their close homologs are key targets for increased understanding of signal transduction in processes associated with human health - immune system signaling, the DNA damage response, osteoclast differentiation, and general cellular growth and differentiation control. These kinases are prototypical of many signaling molecules in that it consists of multiple functional modules, some of which are independently folded structural domains, and these interact both among themselves, and with other ligands, in complex ways which are not readily characterized by conventional structure determination methods. In this proposal, the focus will be on the medium resolution interaction of domains, understanding their role in modification reactions of phosphorylation, and dephosphorylation, the interaction of kinase control with ligands, substrates, adaptors, and the adjacent domains, and examination at high resolution in solution of those which are found amenable in solubility and complexity for complete structural and dynamic characterization. In understanding the role of individual domains and model systems, additional detailed studies will also be undertaken on dynamic properties of multiple domain systems, like Abl SH(32) and Csk(32) by both 15N relaxation methods, and residual dipolar coupling methods; dynamic studies of enzymatically inactivated analogs of down regulated Src; and surveys of the formation of the enzymatic activating complexes of Csk, Src, and their associated phosphatases.
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