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Photosynthetic electron transfers occur through multiple components ranging from small soluble proteins to large integral membrane protein complexes. Co-crystallization of a bacterial photosynthetic electron transfer complex that employs weak hydrophobic interactions was achieved by using high-molar-ratio mixtures of a soluble donor protein (high-potential iron-sulfur protein, HiPIP) with a membrane-embedded acceptor protein (reaction center, RC) at acidic pH. The structure of the co-complex offers a snapshot of a transient bioenergetic event and revealed a molecular basis for thermodynamically unfavorable interprotein electron tunneling. HiPIP binds to the surface of the tetraheme cytochrome subunit in the light-harvesting (LH1) complex-associated RC in close proximity to the low-potential heme-1 group. The binding interface between the two proteins is primarily formed by uncharged residues and is characterized by hydrophobic features. This co-crystal structure provides a model for the detailed study of long-range trans-protein electron tunneling pathways in biological systems., The high potential iron-sulfur (HiPIP) proteins are direct electron donors to the light-harvesting-reaction center complexes (LH1-RC) in photosynthetic β- and γ-Proteobacteria. Here, the authors present the 2.9 Å crystal structure of the HiPIP-bound LH1-RC complex from the thermophilic purple sulfur bacterium Thermochromatium tepidum and discuss mechanistic implications for the electron transfer pathway.

The LH1-RC core complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted interest of many researchers because of its several unique properties, such as increased robustness against environmental hardships and the much red-shifted near-infrared absorption spectrum of the LH1 antenna exciton polarons. The known near-atomic-resolution crystal structure of the complex well supported this attention. Yet several mechanistic aspects of the complex prominence remained to be understood. In this work, samples of the native, Ca2+-containing core complexes were investigated along with those destabilized by Ba2+ substitution, using various spectrally selective steady-state and picosecond time-resolved spectroscopic techniques at physiological and cryogenic temperatures. As a result, the current interpretation of exciton spectra of the complex was significantly clarified. Specifically, by evaluating the homogeneous and inhomogeneous compositions of the spectra...

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q y absorption at 915 nm and enhanced thermostability. Ca2+ can be biosynthetically replaced by Sr2+ in growing cultures of Tch. tepidum. However, the resulting Sr2+-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca2+-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca2+- and Sr2+-cultured cells. In this study, we examined the effects of the biosynthetic Sr2+-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr2+-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr2+-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr2+ substitutes for Ca2+ in the LH1 complex. Limited proteolysis of the native Ca2+-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr2+-substituted LH1-RC complexes in their Ca2+-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Qy absorption band in the range of 870–890 nm, LH1 from the thermophilic bacterium Thermochromatium tepidum displays the Qy band at 915 nm with an enhanced thermostability. These properties are regulated by Ca2+ ions. Substitution of the Ca2+ with other divalent metal ions results in a complex with the Qy band blue-shifted to 880–890 nm and a reduced thermostability. Following the recent publication of the structure of the Ca-bound LH1-reaction center (RC) complex [Niwa, S., et al. (2014) Nature 508, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Qy band at 888 nm. Sixteen Sr2+ and Ba2+ ions are identified in the LH1 complexes. Both Sr2+ and Ba2+ are located at the same positions, and these are clearly different from, though close to, the Ca2+-binding sites. Conformational rearrangement induced by the substitution is limited to the metal-b...

To explore the photoprotection role of multicompositional carotenoid (Car) in photosynthetic purple bacteria, we investigated, by means of triplet excitation profile (TEP) combined with steady-state optical spectroscopies, the core light-harvesting complex-reaction center of a mutant strain of Rhodobacter sphaeroides (m-LH1-RC) at room temperature. TEP spectra revealed that spheroidene and derivative (Spe) preferentially protect bacteriochlorophylls (BChls) of relatively lower site energy by quenching the triplet excitation (3BChl*); however, spirilloxanthin (Spx) does so irrespective to the site energy of BChls. Triplet excitation results showed the triplet excitation energy-transfer (EET) reaction in a timescale of ∼0.5 μs from Spe and derivatives as a major component (∼85%) to Spx as a minor component (∼8%), suggesting the coexistence of different kinds of Cars in the individual LH1 complex. The nonequivalent quenching potency and the triplet EET reaction between Cars constitute the cooperative photopr...

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q

The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αβ-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.

Quinone distributions in the thermophilic purple sulfur bacterium Thermochromatium tepidum have been investigated at different levels of the photosynthetic apparatus. Here we show that, on average, the intracytoplasmic membrane contains 18 ubiquinones (UQ) and 4 menaquinones (MQ) per reaction center (RC). About one-third of the quinones are retained in the light-harvesting–reaction center core complex (LH1–RC) with a similar ratio of UQ to MQ. The numbers of quinones essentially remains unchanged during crystallization of the LH1–RC. There are 1–2 UQ and 1 MQ associated with the RC-only complex in the purified solution sample. Our results suggest that a large proportion of the quinones are confined to the core complex and at least five UQs remain invisible in the current LH1–RC crystal structure.

An image-sensor-based photo receiver for free-space optical communications, especially, ones employing indoor optical wireless LANs, has been developed. The receiver enables a compact and intelligent optical communication system through its two functional modes : image-sensing and communication. We clarified that the diffusion of photo-generated electrons determines the minimum total transimpedance gain of the amplifier and the maximum intensity of optical communication signals. A 50x50-pixel photo receiver with four independent communication-output channels was designed and fabricated with standard 0. 35 μm CMOS technology. To amplify optical data in the communication mode, it has in-pixel trans-impedance amplifiers and column-parallel main amplifiers. Experiments conducted with it showed that data rates of 50Mbps and 30Mbps were possible for wavelengths of 650nm and 830nm, respectively. Transimpedance gain varied from 660kΩ to 4.9MΩ for the data rate of 10Mbps. We also successfully captured images at a frame rate of 50Hz.

Polymers based on silylated p-hydroxy-α-methylstyrene (SiMST) or methacrylic acid (SiMA) were prepared and their application to positive resists for 157nm lithography was studied. These polymers were synthesized by silylation of the copolymer of methacrylonitrile (MAN) and p-hydroxy-α-methylstyrene (HMST) or copolymerization of corresponding monomers. Etch rate of the films using O2 plasma was 12-45nm/min which was lower than that for poly(vinylphenol) film (80nm/min). The incorporation of Si atoms in the polymer side chain strongly enhanced the etch resistance. Changes in a mass of the films during irradiation were studied by in-situ QCM method. Weight loss for SiMA units was larger than that for SiMST units. Dissolution properties of the films in 2.38wt% TMAH aqueous solution were studied and resist pattern profile was simulated.

Digital holographic profilometry using multiwavelength from laser diodes is applied to an inspection of inner surface of straight copper pipe and the detection of artificial defects such as a hole, rust and a scratch in its wall. To obtain the inner surface profile, a cone-shaped mirror attached to a rod having two acrylic spacers is inserted into the pipe and illuminated by the collimated laser beam from the other open end of the pipe. The measurement has been performed by shifting the mirror stepwise along the pipe. Distribution of an optical path length in the alignment is calculated and used to compensate for a distortion in the profile due to a positional misalignment of the mirror. The new algorithm to obtain the positional error is adopted for the compensation process and the shape and positions of defects in the inner wall can be investigated.

A paint drying process is assessed by digital holography. Quantitative analysis using the reconstructed complex amplitudes of the light from the paint surface is performed and the time of tack-free drying under different temperature is investigated.

We propose and demonstrate a new image sensor specialized in an optical wireless LAN (local area network) system. The use of the image sensor brings excellent features to the optical wireless LAN systems; because the image sensor can capture the scene around the communication node or the hub at once like ordinary image sensors, it is easy to implement detection and tracing of the nodes or the hub without any mechanical component to search them. In addition, the image sensors inherently catch the multiple optical signals in parallel by a huge amount of micro photodiodes, which means that they have potentiality of concurrent data acquisition from multiple nodes at the hub. In this paper, we describe a pixel structure to implement two functional modes: a communication (COM) mode to receive temporally modulated optical signals and an image sensor (IS) mode to capture an image in which photodiode operates in an integration mode. The pixel is a fusion of an active pixel sensor and a current amplifier without temporal integration. We fabricated an 8×8-pixel image sensor in a standard 0.8μm BiCMOS technology, and successfully demonstrated position detection of a light source and acquisition of optical serial data when wavelength and frequency of incident optical data were 830nm and 1MHz, respectively. We also show the design of a 50×50-pixel CMOS image sensor with 3-stage main amplifiers.

Digital holographic profilometry using multiwavelength from two laser diodes is applied to the measurement of the inner surface of a straight copper pipe and the detection of artificial defects such as holes, rust, and scratches made on the inner wall. To obtain the inner surface profile, a cone-shaped mirror (CSM) attached to a metal rod having two acrylic spacers is inserted into the pipe and illuminated by the collimated laser beam from the other open end of the pipe. The inspection of the pipe has been performed by shifting the CSM stepwise along the pipe axis. The new algorithm in which a positional misalignment of the CSM can be directly obtained from the experimental height profile can reduce the load for calculation of correcting the distorted experimental height profile. The pipe inspection has been done using the developed images of both the intensity and height profile for the inner wall of the pipe.