Abstract
The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, thereby functioning as a molecular switch. Ras forms a nanocluster on the plasma membrane by modifying lipids in the hypervariable region (HVR) at the C-terminus to exhibit physiological functions. In this study, we demonstrated that chemical modification of cysteine residues in HVR with caged compounds (instead of lipidation) induces multimerization of H-Ras. The sulfhydryl-reactive caged compound, 2-nitrobenzyl bromide (NBB), was stoichiometrically incorporated into the cysteine residue of HVR and induced the formation of the Ras multimer. Light irradiation induced the elimination of the 2-nitrobenzyl group, resulting in the conversion of the multimer to a monomer. SEC-HPLC and small-angle X-ray scattering (SAXS) analysis revealed that H-Ras forms a pentamer. Electron microscopic observation of the multimer showed a circular ring shape, which is consistent with the structure estimated from X-ray scattering. The shape of the multimer may reflect the physiological state of Ras. It was suggested that the multimerization and monomerization of H-Ras were controlled by modification with a caged compound in HVR under light irradiation.
Structural Analysis of Small G-Protein Ras Multimer Induced by Chemical Modification of HVR Domain with Caged Compound