Site-directed mutagenesis, fluorescence, and two-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 sensitivity to the GTPase activating protein (GAP). The signal-transducing activity of the oncogenic Ras protein in PC12 cells was lost upon mutations Y32F and Y40K, but retained upon mutations Y32W and Y40W. These results suggest that residues 32 and 40 are both required to have aromatic 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 sensitivity. By the Y40K mutation, GAP-binding affinity was slightly lowered, while GAP sensitivity was drastically impaired. Therefore, for residues 32 and 40 of Ras, interactions with GAP appear to be different from those with the target of signal transduction in the PC12 cell. As for the Y32W-Ras protein bound with an unhydrolyzable GTP analogue (GMPPNP), the Trp32 fluorescence is appreciably red-shifted, weaker, and more susceptible to KI quenching as compared to that of the GDP-bound form. Two-dimensional NMR spectroscopy with selectively deuterated Ras proteins revealed fewer and weaker nuclear Overhauser effects on the aromatic protons of Trp32 in the GMPPNP-bound form than in the GDP-bound form. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamasaki
K
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
Shirouzu
M
M
Muto
Y
Y
Fujita-Yoshigaki
J
J
Koide
H
H
Ito
Y
Y
Kawai
G
G
Hattori
S
S
Yokoyama
S
S
Nishimura
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)