PROBING THE ROLE OF LOOP-2 IN RAS FUNCTION WITH UNNATURAL AMINO-ACIDS

The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crys tal structures have revealed significant conformational differences in this region between the GTP- and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interactio n with GTPase-activating protein (GAP), suggesting that this region ma y contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, Thr-35, and Ile-36. A Pro-34 --> methanoproline mutant, in which residue 34 is lo cked in the trans conformation, was found to retain high levels of int rinsic and GAP-activated GTPase activity, making unlikely conformation al isomerization at this position. Deletion of a single methyl group f rom Ile (Ile-36 --> norvaline) abolished GAP activation of Ras, reveal ing a remarkable specificity in this protein-protein interaction. Fina lly, replacement of Thr-35 with diastereomeric allo-threonine led to i nactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.