Photo Regulation of Small G-Protein RAS using Photochromic Peptide

Abstract

The small guanine nucleotide binding proteins (G-protein), which are known as a molecular switch is central regulator of cellular signaling pathway. The regulation mechanism of G-protein is well studied at molecular level. Ras is one of the G-protein which have essential role in cellular signal is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). GTP-Ras induced by GEF is active state, whereas GDP-Ras induced by GAP is inactive state. The conformational change induced by GTP binding enables Ras to transduce a signal into downstream through direct interaction with its effectors. Also, mutant Ras known as constitutive active cause excess signaling transduction to downstream effectors, which is pathogenesis of canceration. In this study, we focused on the interaction between Ras and its GEF, Son of Sevenless (SOS) to control Ras function using photochromic compounds. Previously it was shown that the peptide mimicking the αH-helix, Ras binding region of SOS effectively inhibits Ras function competing with native SOS. Therefore, it is expected that photo-induced structural change of the peptide conjugated with photochromic compound enable us to control Ras activity indirectly. We designed and synthesized several SOS αH-Helix peptides which contains two cysteine residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative, ABDM which change its structure reversibly by UV-VIS irradiation. CD and NMR analysis revealed that the modification of the peptide with ABDM stabilizes secondary structure and UV and VIS light irradiations induce secondary structural change reversibly. SOS dependent nucleotide exchange reaction of Ras in the presence of ABDM-SOS peptide were also altered photo-reversibly. Spontaneous cellular uptake of FITC conjugated peptide was observed by confocal microscopy. We also examined the photoreversible ERK pathway inhibition with ABDM-peptide in HeLa cell.