Development of UV Light Irradiation Patterning of Bacteriorhodopsin Thin Films for Biomimetic Functional Devices.

We developed a new patterning method for bacteriorhodopsin (bR) thin films using UV light irradiation. The proton pump function of bR thin films can be deactivated with UV light irradiation. Inactivation of the proton pump function of bR is related to structural changes or photo-bleaching of the retinal in bR using UV light exposure, which was confirmed with absorption and Raman spectroscopy measurements. Utilizing inactivation of the proton pump function with UV light irradiation, we prepared a bR photocell with a stripe-patterned bR thin film and measured its photocurrent response. The new patterning method is applicable to complicated patterning and patterning with a higher spatial resolution, which extends the application of bR thin films as sensor devices.

Enhanced Photocurrent Generation from Bacteriorhodopsin Photocells Using Grating-Structured Transparent Conductive Oxide Electrodes.

We fabricated a grating-structured electrode made of indium-doped zinc oxide (IZO) with a high refractive index (approximately 2) for a bacteriorhodopsin (bR) photocell. We investigated the photocurrent characteristics of the bR photocell and demonstrated that the photocurrent values from the bR/IZO electrode with the grating structure with a grating period of 340 nm were more than 3.5-4 times larger than those without the grating structure. The photocurrent enhancement was attributed to the resonance effect due to light coupling to the grating structure as well as the scattering effect based on the experimental results and analysis using the photonic band structure determined using finite-difference time-domain (FDTD) simulations. The refractive index of the bR film in electrolyte solution (1.40) used in the FDTD simulations was estimated by analyzing the extinction peak wavelength of 20-nm gold colloids in the bR film. Our results indicate that the grating- or photonic-crystal-structured transparent conductive oxide (TCO) electrodes can increase the light use efficiency of various bR devices such as artificial photosynthetic devices, solar cells, and light-sensing devices.

Isolation and characterization of a virus (CvV-BW1) that infects symbiotic algae of Paramecium bursaria in Lake Biwa, Japan.

BACKGROUND: We performed an environmental study of viruses infecting the symbiotic single-celled algae of Paramecium bursaria (Paramecium bursaria Chlorella virus, PBCV) in Lake Biwa, the largest lake in Japan. The viruses detected were all Chlorella variabilis virus (CvV = NC64A virus). One of them, designated CvV-BW1, was subjected to further characterization. RESULTS: CvV-BW1 formed small plaques and had a linear DNA genome of 370 kb, as judged by pulsed-field gel electrophoresis. Restriction analysis indicated that CvV-BW1 DNA belongs to group H, one of the most resistant groups among CvV DNAs. Based on a phylogenetic tree constructed using the dnapol gene, CvV was classified into two clades, A and B. CvV-BW1 belonged to clade B, in contrast to all previously identified virus strains of group H that belonged to clade A. CONCLUSIONS: We conclude that CvV-BW1 composes a distinct species within C. variabilis virus.

Effects of the disappearance of one charge on ultrafast fluorescence dynamics of the FMN binding protein.

Crystal structures of E13T (Glu13 was replaced by Thr13) and E13Q (Glu13 was replaced by Gln13) FMN binding proteins (FMN-bp) from Desulfovibrio vulgaris, strain Miyazaki F, were determined by the X-ray diffraction method. Geometrical factors related to photoinduced electron transfer from Trp32, Tyr35, and Trp106 to the excited isoalloxazine (Iso*) were compared among the three forms of FMN-bp. The rate of ET is considered to be fastest from Trp32 to Iso* in FMN-bp and then from Tyr35 and Trp106. The distances between Iso and Trp32 did not change appreciably (0.705-0.712 nm) among WT, E13T, and E13Q FMN-bps, though the distances between Iso and Tyr35 or Trp106 became a little shorter by ca. 0.01 nm in both mutated FMN-bps. The distances between the residue at 13 and the ET donors or acceptor in the mutated proteins, however, changed markedly, compared to WT. Hydrogen bonding pairs and distances between Iso and surrounding amino acids were not modified when Glu13 was replaced by Thr13 or Gln13. Effects of elimination of ionic charge at Glu13 on the ultrafast fluorescence dynamics in E13T and E13Q were investigated comparing to WT, by means of a fluorescence up-conversion method. Fluorescence lifetimes were tau(1) = 107 fs (alpha(1) = 0.86), tau(2) = 475 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.02) in E13T and tau(1) = 134 fs (alpha(1) = 0.85), alpha(2) = 746 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.03) in E13Q, which are compared to the reported lifetimes in WT, tau(1) = 168 fs (alpha(1) = 0.95) and alpha(2) = 1.4 ps (alpha(2) = 0.05). Average lifetimes (tau(AV) = Sigma(i=1)(2or3)alpha(i)tau(i)) were 0.75 ps in E13T, 1.10 ps in E13Q, and 0.23 ps in WT, which implies that tau(AV) was 3.3 times longer in E13T and 4.8 times longer in E13Q, compared to WT. The ultrafast fluorescence dynamics of WT did not change when solvent changed from H(2)O to D(2)O. Static ET rates (inverse of average lifetimes) were analyzed with static structures of the three systems of FMN-bp. Net electrostatic (ES) energies of Iso and Trp32, on which ET rates depend, were 0.0263 eV in WT, 0.322 eV in E13T, and 0.412 eV in E13Q. The calculated ET rates were in excellent agreement with the observed ones in all systems.

Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family.

The crystal structure of flavoredoxin from Desulfovibrio vulgaris Miyazaki F was determined at 1.05 A resolution and its ferric reductase activity was examined. The aim was to elucidate whether flavoredoxin has structural similarity to ferric reductase and ferric reductase activity, based on the sequence similarity to ferric reductase from Archaeoglobus fulgidus. As expected, flavoredoxin shared a common overall structure with A. fulgidus ferric reductase and displayed weak ferric reductase and flavin reductase activities; however, flavoredoxin contains two FMN molecules per dimer, unlike A. fulgidus ferric reductase, which has only one FMN molecule per dimer. Compared with A. fulgidus ferric reductase, flavoredoxin forms three additional hydrogen bonds and has a significantly smaller solvent-accessible surface area. These observations explain the higher affinity of flavoredoxin for FMN. Unexpectedly, an electron-density map indicated the presence of a Mes molecule on the re-side of the isoalloxazine ring of FMN, and that two zinc ions are bound to the two cysteine residues, Cys39 and Cys40, adjacent to FMN. These two cysteine residues are close to one of the putative ferric ion binding sites of ferric reductase. Based on their structural similarities, we conclude that the corresponding site of ferric reductase is the most plausible site for ferric ion binding. Comparing the structures with related flavin proteins revealed key structural features regarding the discrimination of function (ferric ion or flavin reduction) and a unique electron transport system.