Conformational Change Of Hvr Domain Of Small Gtpase Ras Reflecting Physiological Function

The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switching mechanisms utilizing conformational changes in the nucleotide-binding motifs have been well studied at the molecular level. H-Ras has the hypervariable region (HVR) at the C-terminal. It is known that HVR is modified by farnesylation and palmitoylation. The lipid modification enable H-Ras bind to membrane to act physiological role. We have previously shown that chemical modification of the cysteine residues in the HVR of Ras with bulky hydrophobic SH group specific reagents induced multimerization of Ras. The HVR domain is believed to perform physiologically important roles. Therefore, the multimerization phenomenon may reflect the physiological function of Ras. In this study, we analyzed conformation of the HVR domain in the Ras multimer by synchrotron X ray scattering and fluorescent probes. Small angle X ray scattering analysis suggested that the Ras modified with fluorescent probes forms pentamer. Interfaces between subunits of the pentamer was examined by the fluorescence quenching method. The results suggested that HVR region is located in the interface. Furthermore, we also analyzed the conformation of HVR domain by FRET. The FRET efficiencies from the tryptophan in HVR domain to the fluorophore of NBD-GTP or NBD-GDP trapped in the GTP binding site indicated that HVR domain of Ras-GDP locates closer to catalytic domain than Ras-GTP.