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 crystal 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 interaction with GTPase-activating protein (GAP), suggesting that this region may 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 locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Ile (Ile-36-->norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.