The objective of this project is to obtain high resolution solution structures of proteins containing the SH (Src-Homology) domains 2 and 3 using multiple dimensional NMR. These domains are found in many proteins that are involved in signal transducing pathways and have been implicated in the inter- and intra-molecular regulation of kinase and other enzyme activities and in the oncogenic potential of these proteins. As well, one SH2 domain has been demonstrated to be sufficient for the high affinity binding of tyrosyl-phosporylated proteins and and another SH3 domain may bind actin filaments of the cellular cytoskeleton. This study is proposed in order to generate the first three dimensional structures of these novel domains and to identify their intramolecular interactions and phosphotyrosyl-containing protein binding sites. The immediate goal is to proceed from circular dichroism and two-dimensional NMR studies already completed with unlabelled protein to a complete assignment and secondary structure analysis of a N15 labelled 107 residue protein containing the 80- amino acid SH2 domain from c-abl protein. A high resolution tertiary structure determination using interproton distance constraints and torsion angles from C-13 and N-15 labeled protein would be generated. Amino acid residues involved in binding phosphotyrosyl containing substrate would be identified by NMR. Since the transforming activity of several oncogenes has been traced to mutations in their SH2 domains, the three dimensional structure of the SH2 domain of the c-abl proto-oncogene should provide a structural explanation for their oncogenic activity, and may suggest rational routes to therapies for related conditions.
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