Seigo Iwata

Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes.

Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.

Photocontrol of the GTPase activity of the small G protein K-Ras by using an azobenzene derivative

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans forms upon exposure to ultraviolet and visible light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the nucleotide-binding motif of Ras to regulate the GTPase activity by photoirradiation. We prepared four Ras mutants (G12C, Y32C, I36C, and Y64C) that have a single reactive cysteine residue in the nucleotide-binding motif. PAM was stoichiometrically incorporated into the cysteine residue of the mutants. The PAM-modified mutants exhibited reversible alterations in GTPase activity, nucleotide exchange rate, and interaction between guanine nucleotide exchange factor and Ras, accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. The results suggest that incorporation of photochromic molecules into its nucleotide-binding motif enables photoreversible control of the function of the small G protein Ras.

Photoregulation of Small G Protein Kras using Photochromic Molecules

Ras is one of small G-proteins known as a molecular switch mediating cellular signalling. Switching ON state of Ras is induced by exchange of bound GDP for GTP and OFF state is by hydrolysis of GTP to GDP. Interestingly, the core nucleotide-binding motif of Ras is considerably conserved with the ATP driven motor proteins, myosin and kinesin. Therefore, it is believed that these bio-molecular machines share common molecular mechanism utilizing nucleotide hydrolysis cycle. Previously, we have incorporated photochromic molecules, 4-phenylazophenyl maleimide (PAM), into the functional site of kinesin as a photo-switching device and succeeded to regulate kinesin ATPase activities reversibly upon visible light (VIS) and ultra-violet (UV) light irradiation. Therefore, it is expected that Ras can be also regulated using photochromic molecules.In this study, we performed basic study to control the function of Ras reversibly using photochromic molecules upon VIS and UV light irradiations. First, in order to monitor the exchange of bound GDP for GTP, we synthesized a new fluorescent GTP analogue, NBD-GTP and NBD-GDP, which change their fluorescent intensity during their binding to Ras. Second, we synthesized a new photochromic molecule, iodoacetyl spiropyran (IASP) and iodoacetyl fulgimide (IAFI) that are incorporated into cysteine residue specifically. And the GTPase activity of Ras was monitored by the quantitative analysis of GTP and GDP in the active site of Ras using reverse phase column chromatography on HPLC. We have designed three kinds of Ras mutants Y32C, I36C, and Y64C. The mutants were prepared using E.coli.expression system and modified with PAM and IASP stoichiometrically. It was suggested that the GTPase activities of the Ras mutants modified with PAM were reversibly alternated upon VIS and UV light irradiations. The Ras mutants modified with IASP and IAFI were also examined.