Photocontrol of Small G-Protein H-RAS Multimer Formation using Caged Compounds

Abstract

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, in which three cysteine residues are located. It is known that HVR is the site of lipid modification; farnesylation and palmitoylation. Lipid modification enable H-Ras bind to membrane to act physiological role. Recently we have observed that the chemical modification of the cysteine residues in the HVR with hydrophobic thiol reactive reagents induces multimer formation of H-Ras. In this study, utilizing the phenomenon, we attempted to control the transition from multimer to monomer of H-Ras by light irradiation. We employed thiol reactive caged-compound, 2-Nitrobenzyl bromide (NBB). NBB was incorporated into the cysteine residues in HVR stoichiometrically. The modification of H-Ras with NBB induced formation of multimer. We monitored the transition between the monomer and multimer by Size-exclusion chromatography coupled with high-performance liquid chromatography (SEC-HPLC). Light irradiation at 400 nm induced elimination of 2-Nitrobenzyl group, resulting in conversion of multimer to monomer. The elution pattern of the multimer on SEC-HPLC indicated the multimer is composed of five Ras molecules. Small angle X-ray scattering (SAXS) analysis of the multimer also suggested that the NBB modified Ras formed the pentamer. The global shape of the multimer was deduced from the data of SAXS. We prepared H-Ras mutants which intrinsic cysteine residues in HVR were substituted with serine in order to identify which cysteine residue is essential for the multimerization. Incorporations of NBB into more than two residues among the three cysteines were shown to be necessary for multimerization.