Photoregulation of Small G-Proteins using Photochromic Molecules

Abstract

The small guanine nucleotide binding proteins (G-protein), which are known as a molecular switch, is a central regulator of cellular signal transduction processes leading to transcription, cell cycle progression, growth, migration, cytoskeletal changes, apoptosis, cell survival, and senescence. The switching mechanism has been well studied at the molecular level. It is known that the G-proteins are activated in the GTP bound state and inactivated in the GDP bound state. The conformational change induced by GTP binding enables Ras to transduce a signal downstream through direct interaction with its effectors. The switching of G-protein is controlled by Guanine nucleotide Exchange Factor (GEF) and GTPase Activating Protein (GAP). Firstly, we attempted to control G-proteins RhoA and Ras photo-reversibly by introducing photochromic molecules into the functional regions of G-protein. We prepared RhoA and H-Ras mutants which have a single cysteine residue at the functional site and modified with photochromic azobenzene and spiropyran derivatives. GTPase and nucleotide exchange rate for some of the photochromic molecules modified RhoA and Ras were photo-reversibly altered. We also examined photo-regulated interaction of RhoA modified with photochromic molecule with its target kinase ROCK2. Secondly, we introduce photochromic molecules into the regulatory factor GEF. It is known that αH-helix region of SOS (GEF) is located at the interface to Ras. We designed and synthesized SOS αH-helix peptide which contains two cysteine residues at the cross-linkable positions with bifunctional azobenzene derivative. The peptide was cross-linked with azobenzene di-maleimide intramolecularly. Photo-reversible interaction of the ABDM modified peptide with Ras was examined.