Biomedical subjects

Kenichi Ataka

Publications and source records attributed to Kenichi Ataka.

4 recordsLinked to original sources

PubMed2004

Functional vibrational spectroscopy of a cytochrome c monolayer: SEIDAS probes the interaction with different surface-modified electrodes.

Electrochemically induced infrared difference spectra of cytochrome c on various chemically modified electrodes (CMEs) are recorded by exploiting the surface-enhancement exerted by a granular gold film. We have recently developed surface-enhanced infrared difference absorption spectroscopy (SEIDAS), which provides acute sensitivity to observe the minute enzymatic change of a protein on the level of a monolayer. By these means, we demonstrate that the relative band intensities in the potential-induced difference spectra of adsorbed cytochrome c are significantly dependent on the type of CME used (mercaptopropionic acid, mercaptoethanol, 4,4'-dithiodipyridine, or L-cysteine). These differences are attributed to the altered interaction of cytochrome c with the headgroup of the various CMEs leading to variations in surface orientation and relative distance from the surface. Nevertheless, the peak positions of the observed bands are identical among the CMEs employed. This implies that the internal conformational changes induced by the redox reaction of the adsorbed cytochrome c are not disturbed by the interaction with the CME and that full functionality of the protein is retained. Finally, we critically discuss our results within the framework of the different models for cytochrome c adsorption on CMEs.

Crystallography, X-Ray↗

PubMed2004

The molecular mechanism of membrane proteins probed by evanescent infrared waves.

The catalytic action of membrane proteins is vital to many cellular processes. Yet the molecular mechanisms remain poorly understood. We describe here the technique of evanescent infrared difference spectroscopy as a tool to decipher the structural changes associated with the enzymatic action of membrane proteins. Functional changes as minute as the protonation state of individual amino acid side chains can be observed and linked to interactions with a ligand, agonist, effector, or redox partner.

Bacteriorhodopsins↗

PubMed2003

Electrochemically induced surface-enhanced infrared difference absorption (SEIDA) spectroscopy of a protein monolayer.

Functional infrared difference spectra of a protein monolayer are recorded by exploiting the surface enhancement exerted by a modified gold surface. The noble metal is used as the working electrode to induce redox-linked structural changes in the single layer of cytochrome c. The observed differences in the relative band intensities as compared to the difference spectrum of cytochrome c in solution are attributed to orientational effects of the adsorbed protein.

Animals↗

PubMed2002

Proteorhodopsin is a light-driven proton pump with variable vectoriality.

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

Animals↗