SITE-DIRECTED MUTAGENESIS, FLUORESCENCE, AND 2-DIMENSIONAL NMR-STUDIES ON MICROENVIRONMENTS OF EFFECTOR REGION AROMATIC RESIDUES OF HUMAN C-HA-RAS PROTEIN

The Tyr residues in positions 32 and 40 of human c-Ha-Ras protein were replaced by site-directed mutagenesis (Y32F, Y32W, Y40K, and Y40W) to examine their roles in the signal-transducing activity and the sensit ivity to the GTPase activating protein (GAP). The signal-transducing a ctivity of the oncogenic Ras protein in PC12 cells was lost upon mutat ions Y32F and Y40K, but retained upon mutations Y32W and Y40W. These r esults suggest that residues 32 and 40 are both required to have aroma tic groups and residue 32 is further required to have a hydrogen donor . On the other hand, three mutations (Y32F, Y32W, and Y40W) caused no appreciable reduction in either GAP-binding affinity or GAP sensitivit y. By the Y40K mutation, GAP-binding affinity was slightly lowered, wh ile GAP sensitivity was drastically impaired. Therefore, for residues 32 and 40 of Ras, interactions with GAP appear to be different from th ose with the target of signal transduction in the PC12 cell. As for th e Y32W-Ras protein bound with an unhydrolyzable GTP analogue (GMPPNP), the Trp32 fluorescence is appreciably red-shifted, weaker, and more s usceptible to KI quenching as compared to that of the GDP-bound form. Two-dimensional NMR spectroscopy with selectively deuterated Ras prote ins revealed fewer and weaker nuclear Overhauser effects on the aromat ic protons of Trp32 in the GMPPNP-bound form than in the GDP-bound for m. This indicates that the side chain of Trp32 is more exposed to the solvent in the GMPPNP-bound form than in the GDP-bound form. In contra st, fluorescence and NMR analyses for the Y40W-Ras protein indicate th at the microenvironment of Trp40 is only slightly affected upon GDP-GM PPNP exchange. Accordingly, the aromatic residue in position 32, rathe r than that in position 40 of the Ras protein, becomes exposed to the surface of the protein upon GDP-GTP exchange, allowing hydrogen bondin g with a target molecule in the signal-transduction pathway.