Regulated inter- and intramolecular protein interactions play central roles in signaling events that control lymphocytes responses. The Itk protein tyrosine kinase, a member of the Tec family, plays an important role in T cell development and activation. Previous work from this investigators postdoctoral studies revealed a novel intramolecular interaction involving proline residues and the SH3 domain in Itk. Here, the investigator proposes to extend those studies, based on preliminary data, that suggest additional regulatory intramolecular interactions which depend upon the TH and SH2 domain of Itk. High resolution multidimensional nuclear magnetic resonance (NMR) techniques will be used to explore this intramolecular interaction. Structures and interactions of protein fragments lacking the catalytic domain of the N-terminus of Itk will be studied to investigate the intramolecular associations that might regulate substrate binding and catalytic function in the intact protein. These studies may provide new knowledge regarding signal transduction by T and B cell antigen receptors. Furthermore, these studies may broaden the application of multi- dimensional NMR techniques to the study of larger multi-domain protein systems. Novel techniques combining recombinant protein expression and chemical ligation to generate proteins in which a single domain is isotopically labeled are proposed. This technology will not only provide detailed information about intramolecular interactions in Itk, but will be broadly applicable to the study of intramolecular interactions in other protein with complex domain structure.
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