This proposal will extend the knowledge of the solution structure of a large protein tyrosine kinase, Abelson and its complexes in intracellular signal transduction, using both established NMR and molecular biology approaches and new technologies. These new technologies include those developed by the PI such as, segmental labeling using expressed protein ligation, domain structures with consolidated ligands and direct determination of segmental motion of multidomain proteins from relaxation studies. A long-term objective is to understand the molecular basis of the action of Abl, which in connection with a chromosomal translocation is the causative agent in chronic myologeneous leukemia. Abl is also 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 this proposal, the focus is on the DNA binding domain, the interaction of tyrosine kinase control with other ligands, substrates, and the adjacent SH2 and SH3 domains, and comparison systems for the letter, including Csk and Lck, simpler protein tyrosine kinases, which are now more practical for complete structural and dynamic characterization. There are three structural biology aims and three methodology aims: 1) to investigate the structure of the DNA binding domain of Abl, to determine the structural basis of selectivity. 2) To investigate the interdomain interactions of Src Homology kinase, SH2 and SH3 domains in Abl, and in related PTKs CSK, Hck, Lck and their mechanism of control of enzymatic activity. 3) To investigate the apoptosis-related structural biology of possible Abl downstream effectors, specifically Bid, its truncated forms, and BH3-helix mimics. 4) Segmental labeling of target proteins for rapid structure and dynamics determination. 5) Orientation dependent characterization of structure and dynamics from relaxation, and residual dipolar coupling. 6) Obtain highly accurate structures and dynamics of ligated domains and multidomain constructs using improved NMR methods and ligands, with a long-term view to improving the process of developing investigational and therapeutic agents.
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