掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.bbabio.2021.148471

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -state cycle, leading to the generation of di-oxygen, protons and electrons. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

DOI： 10.1107/S2052252521002177

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出版者・発行元：Cold Spring Harbor Laboratory  

Abstract

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

DOI： 10.1101/2020.10.18.344648

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Photosynthetic light-harvesting complexes (LHCs) play a pivotal role in collecting solar energy for photochemical reactions in photosynthesis. One of the major LHCs are fucoxanthin chlorophyll a/c-binding proteins (FCPs) present in diatoms, a group of organisms having important contribution to the global carbon cycle. Here, we report a 2.40-Å resolution structure of the diatom photosystem I (PSI)-FCPI supercomplex by cryo-electron microscopy. The supercomplex is composed of 16 different FCPI subunits surrounding a monomeric PSI core. Each FCPI subunit showed different protein structures with different pigment contents and binding sites, and they form a complicated pigment-protein network together with the PSI core to harvest and transfer the light energy efficiently. In addition, two unique, previously unidentified subunits were found in the PSI core. The structure provides numerous insights into not only the light-harvesting strategy in diatom PSI-FCPI but also evolutionary dynamics of light harvesters among oxyphototrophs.

DOI： 10.1038/s41467-020-16324-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Iron-stress induced protein A (IsiA) is a chlorophyll-binding membrane-spanning protein in photosynthetic prokaryote cyanobacteria, and is associated with photosystem I (PSI) trimer cores, but its structural and functional significance in light harvesting remains unclear. Here we report a 2.7-Å resolution cryo-electron microscopic structure of a supercomplex between PSI core trimer and IsiA from a thermophilic cyanobacterium Thermosynechococcus vulcanus. The structure showed that 18 IsiA subunits form a closed ring surrounding a PSI trimer core. Detailed arrangement of pigments within the supercomplex, as well as molecular interactions between PSI and IsiA and among IsiAs, were resolved. Time-resolved fluorescence spectra of the PSI-IsiA supercomplex showed clear excitation-energy transfer from IsiA to PSI, strongly indicating that IsiA functions as an energy donor, but not an energy quencher, in the supercomplex. These structural and spectroscopic findings provide important insights into the excitation-energy-transfer and subunit assembly mechanisms in the PSI-IsiA supercomplex.

DOI： 10.1038/s42003-020-0949-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. SCOPE OF REVIEW: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. MAJOR CONCLUSIONS: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. GENERAL SIGNIFICANCE: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules.

DOI： 10.1016/j.bbagen.2019.129466

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chlorophylls (Chl) play pivotal roles in energy capture, transfer and charge separation in photosynthesis. Among Chls functioning in oxygenic photosynthesis, Chl f is the most red-shifted type first found in a cyanobacterium Halomicronema hongdechloris. The location and function of Chl f in photosystems are not clear. Here we analyzed the high-resolution structures of photosystem I (PSI) core from H. hongdechloris grown under white or far-red light by cryo-electron microscopy. The structure showed that, far-red PSI binds 83 Chl a and 7 Chl f, and Chl f are associated at the periphery of PSI but not in the electron transfer chain. The appearance of Chl f is well correlated with the expression of PSI genes induced under far-red light. These results indicate that Chl f functions to harvest the far-red light and enhance uphill energy transfer, and changes in the gene sequences are essential for the binding of Chl f.

DOI： 10.1038/s41467-019-13898-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jpcb.9b10154

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Photosystem I (PSI) functions to harvest light energy for conversion into chemical energy. The organisation of PSI is variable depending on the species of organism. Here we report the structure of a tetrameric PSI core isolated from a cyanobacterium, Anabaena sp. PCC 7120, analysed by single-particle cryo-electron microscopy (cryo-EM) at 3.3 Å resolution. The PSI tetramer has a C2 symmetry and is organised in a dimer of dimers form. The structure reveals interactions at the dimer-dimer interface and the existence of characteristic pigment orientations and inter-pigment distances within the dimer units that are important for unique excitation energy transfer. In particular, characteristic residues of PsaL are identified to be responsible for the formation of the tetramer. Time-resolved fluorescence analyses showed that the PSI tetramer has an enhanced excitation-energy quenching. These structural and spectroscopic findings provide insights into the physiological significance of the PSI tetramer and evolutionary changes of the PSI organisations.

DOI： 10.1038/s41467-019-12942-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/science.aax6998

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cplett.2019.06.026

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41477-019-0477-x

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その他リンク： http://www.nature.com/articles/s41477-019-0477-x

DOI： 10.1038/s41477-019-0438-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Diatoms are dominant phytoplankton in aquatic environments and have unique light-harvesting apparatus, fucoxanthin chlorophyll a/c-binding protein (FCP). Diatom photosystem I (PSI) interacts with specific FCPs (FCPI); however, it remains unclear how PSI cores receive excitation energy from FCPI. To analyze the energy transfer dynamics, it is necessary to isolate both PSI cores and PSI-FCPI complexes. In this study, we prepared three PSI complexes, which are PSI-FCPI membrane fragments, detergent-solubilized PSI-FCPI supercomplexes and PSI core-like complexes, from the marine centric diatom, Chaetoceros gracilis, and examined their biochemical properties. Both the PSI-FCPI membrane fragments and supercomplexes showed similar subunit compositions including FCPI, whereas the PSI complexes were devoid of most FCPI subunits. The purity and homogeneity of the two detergent-solubilized PSI preparations were verified by clear-native PAGE and electron microscopy. The difference of pigment contents among the three PSI samples was shown by absorption spectra at 77 K. The intensity in the whole spectrum of PSI-FCPI membranes was much higher than those of the other two complexes, while the spectral shape of PSI complexes was similar to that of cyanobacterial PSI core complexes. 77-K fluorescence spectra of the three PSI preparations exhibited different spectral shapes, especially peak positions and band widths. Based on these observations, we discuss the merits of three PSI preparations for evaluating excitation energy dynamics in diatom PSI-FCPI complexes.

DOI： 10.1007/s11120-018-0576-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA118.004304

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society  

Photosynthetic water oxidation is performed in photosystem II (PSII) through a light-driven cycle of intermediates called S states (S0-S4) at the water oxidizing center. Time-resolved serial femtosecond crystallography (SFX) has recently been applied to the microcrystals of PSII to obtain the structural information on these intermediates. However, it remains unanswered whether the reactions efficiently proceed throughout the S-state cycle retaining the native structures of the intermediates in PSII crystals. We investigated the water oxidation reactions in the PSII microcrystals using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. In comparison with the FTIR spectra in solution, it was shown that all of the metastable intermediates in the microcrystals retained their native structures, and the efficiencies of the S-state transitions remained relatively high, although those of the S2 → S3 and S3 → S0 transitions were slightly lowered possibly due to some restriction of water movement in the crystals.

DOI： 10.1021/acs.jpclett.8b00638

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier B.V.  

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn4CaO5. VTVH MCD spectra taken in the S2 state provide a clear g = 2, S = 1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749 nm, negative (−) at 773 nm and (+) at 808 nm are assigned as 4A → 2E spin-flips within the d3 configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S3 exhibits a more intense (−) MCD peak at 764 nm and has a stronger MCD saturation characteristic. This S3 MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S2 EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.

DOI： 10.1016/j.bbabio.2017.10.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Large-scale QM/MM calculations were performed to elucidate an optimized geometrical structure of a CaMn4O5 cluster with and without water insertion in the S-3 state of the oxygen evolving complex (OEC) of photosystem II (PSII). The left (L)-opened structure was found to be stable under the assumption of no hydroxide anion insertion in the S-3 state, whereas the right (R)-opened structure became more stable if one water molecule is inserted to the Mn4Ca cluster. The optimized Mn-a(4)-Mn-d(1) distance determined by QM/MM was about 5.0 angstrom for the S-3 structure without an inserted hydroxide anion, but this is elongated by 0.2-0.3 angstrom after insertion. These computational results are discussed in relation to the possible mechanisms of O-O bond formation in water oxidation by the OEC of PSII.

DOI： 10.1039/c6fd00230g

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)(1-3). The structure of PSII has been analysed at 1.9 angstrom resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, `distorted-chair' form(4). This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the `radiation damage-free'(5) structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 angstrom using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 angstrom from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique mu 4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 angstrom between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously(6,7.)

DOI： 10.1038/nature21400

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The minor outer capsid protein P2 of Rice dwarf virus (RDV), a member of the genus Phytoreovirus in the family Reoviridae, is essential for viral cell entry. Here, we clarified the structure of P2 and the interactions to host insect cells. Negative stain electron microscopy (EM) showed that P2 proteins are monomeric and flexible L-shaped filamentous structures of similar to 20 nm in length. Cryo-EM structure revealed the spatial arrangement of P2 in the capsid, which was prescribed by the characteristic virion structure. The P2 proteins were visualized as partial rod-shaped structures of similar to 10 nm in length in the cryo-EM map and accommodated in crevasses on the viral surface around icosahedral 5-fold axes with hydrophobic interactions. The remaining disordered region of P2 assumed to be extended to the radial direction towards exterior. Electron tomography clearly showed that RDV particles were away from the cellular membrane at a uniform distance and several spike-like densities, probably corresponding to P2, connecting a viral particle to the host cellular membrane during cell entry. By combining the in vitro and in vivo structural information, we could gain new insights into the detailed mechanism of the cell entry of RDV.

DOI： 10.1093/jb/mvv092

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

Oxygenic photosynthesis synthesizes sugars from water and carbon dioxide using light energy from the sun, thereby converts light energy into chemical energy and provides oxygen for aerobic life on the earth. The light-harvesting, electron transfer, and water-splitting reactions of photosynthesis are catalyzed by two large membrane-protein complexes photosystem II (PSII) and photosystem I (PSI). Through high-resolution crystal structural analysis by synchrotron X-rays as well as femtosecond X-ray free electron lasers, the mechanisms of these reactions have become understandable at the atomic level. Here we review the recent progresses in analyzing the structures of PSII and PSI as well as their functional implications.

DOI： 10.2142/biophys.56.079

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その他リンク： https://jlc.jst.go.jp/DN/JLC/20021928091?from=CiNii

掲載種別：研究論文（学術雑誌）   出版者・発行元：International Union of Crystallography ({IUCr})  

DOI： 10.1107/s2053273315099751

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

Several key concepts and geometrical rules for the Mn-Mn and Mn-O distances of the CaMn4O5 cluster in the oxygen evolving complex (OEC) of photosystem II (PSII) by previous and present theoretical calculations were examined for a clear understanding of the damage-free Si structure revealed by X-ray free electron laser (XFEL). A simple equation to estimate the Mn-a-Mn-b distance in relation to the Mn-a-O(5) distance was derived taking into consideration the Jahn-Teller (JT) effects for Mn centers, indicating that the XFEL structure is regarded as a slightly right-elongated quasi-central structure in contradiction to a right-opened structure proposed by the EXAFS measurements. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cplett.2015.03.033

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The structure of the oxygen evolving complex (OEC) of photosystem II (PSII) was recently analyzed by crystallography at 1.95 angstrom resolution using X-ray free electron laser (XFEL), but the positions of hydrogen atoms were not determined. We have examined the XFEL structure theoretically under the assumption off our protonation cases. The spin densities obtained by the high-spin UB3LYP revealed that a partial internal reduction of the high-valent Mn ions in the CaMn4O4X(H2O)(3)Y cluster occurs for the O-(5)(=X) = O2- case, entailing its protonation (X = OH-) in the XFEL structure. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cplett.2015.01.030

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Visible/UV absorption in PS II core complexes is dominated by the chl-a absorptions, which extend to similar to 700 nm. A broad 700-730 nm PS II core complex absorption in spinach has been assigned [1] to a charge transfer excitation between Chl(D1) and Chl(D2). Emission from this state, which peaks at 780 nm, has been seen [2] for both plant and cyanobacterial samples. We show that Thermosynechococcus vulcanus PS II core complexes have parallel absorbance in the 700-730 nm region and similar photochemical behaviour to that seen in spinach. This establishes the low energy charge transfer state as intrinsic to the native PS II reaction centre. High-sensitivity MCD measurements made in the 700-1700 nm region reveal additional electronic excitations at similar to 770 nm and similar to 1550 nm. The temperature and field dependence of MCD spectra establish that the system peaking near 1550 nm is a heme-to-Fe(III) charge transfer excitation. These transitions have not previously been observed for cyt b(559) or cyt C-550. The distinctive characteristics of the MCD signals seen at 770 nm allow us to assign absorption in this region to a d(z)(2) -&gt; d(x2) (-) (y2) transition of Mn(III) in the Ca-Mn4O5 cluster of the oxygen evolving centre. Current measurements were performed in the S-1 state. Detailed analyses of this spectral region, especially in higher S states, promise to provide a new window on models of water oxidation. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2014.11.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 angstrom resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment(1). However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation(2), and slight differences were found in the Mn-Mn distances determined by XRD1, EXAFS(3-7) and theoretical studies(8-14). Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S-1 state at a resolution of 1.95 angstroms using femtosecond X-ray pulses of the SPring-8 angstrom compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1-0.2 angstroms. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.

DOI： 10.1038/nature13991

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：HUMANA PRESS INC  

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the mesophilic fungi that can produce high levels of cellulose-related enzymes and are expected to be used for the degradation of polysaccharide biomass. In silico analysis of the genome sequence of T. cellulolyticus detected seven open reading frames (ORFs) showing homology to xylanases from glycoside hydrolase (GH) family 11. The gene encoding the GH11 xylanase C (TcXylC) with the highest activity was used for overproduction and purification of the recombinant enzyme, permitting solving of the crystal structure to a resolution of 1.98 . In the asymmetric unit, two kinds of the crystal structures of the xylanase were identified. The main structure of the protein showed a beta-jelly roll structure. We hypothesize that one of the two structures represents the open form and the other shows the close form. The changing of the flexible region between the two structures is presumed to induce and accelerate the enzyme reaction. The specificity of xylanase toward the branched xylan is discussed in the context of this structural data and by comparison with the other published structures of xylanases.

DOI： 10.1007/s12010-014-1130-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus ostreatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disulfide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic potential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein.

DOI： 10.1248/bpb.b13-00929

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Routinely prepared PS II core samples are often contaminated by a significant (similar to 1-5%) fraction of PSI, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700-730 nm) as PS I (Hughes et al. 2006). When contamination levels are less than similar to 1%, it becomes difficult to quantify the PS I related components by gelbased, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700 nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus volcanos. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780 nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2013.09.008

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.54.S195_1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

It is well known that viruses utilize the host cellular systems for their infection and replication processes. However, the molecular mechanisms underlying these processes are poorly understood for most viruses. To understand these molecular mechanisms, it is essential to observe the viral and virus-related structures and analyse their molecular interactions within a cellular context. Cryo-electron microscopy and tomography offer the potential to observe macromolecular structures and to analyse their molecular interactions within the cell. Here, using cryo-electron microscopy and tomography, the structures of Rice dwarf virus are reported within fully hydrated insect vector cells grown on electron microscopy grids towards revealing the viral infection and replication mechanisms.

DOI： 10.1107/S090904951302219X

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC GENERAL MICROBIOLOGY  

Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a persistent propagative manner. In this study, sequential infection of RRSV in the internal organs of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy. RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary glands of infected insects and contained the non-structural protein Pns10 of RRSV, which appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in non-host insect cells following expression of Pns10 in a baculovirus system, suggesting that the viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited such viroplasm formation, preventing efficient virus infection and spread in its insect vectors. These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication in the vector insect.

DOI： 10.1099/vir.0.042424-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wildtype rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae. (C) 2011 Elsevier B. V. All rights reserved.

DOI： 10.1016/j.jbiotec.2011.12.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN INT RESEARCH CENTER AGRICULTURAL SCIENCES  

Antibodies (immunoglobulin G (IgGs)) from antisera raised against viral particles that had been dissociated by treatment with SDS and intact particles of Rice dwarf virus (RDV) were studied for their ability to prevent viral infection of vector cells in monolayers in vitro. Even though IgGs raised against dissociated virus had a higher titer than those raised against intact viruses in an analysis of viral proteins on Western blots, they did not neutralize RDV. Conversely, IgGs raised against intact RDV effectively neutralized viral infectivity. Electron microscopic observation of the aggregation of RDV particles after incubation with IgGs raised against intact R DV, but no aggregation of R DV particles after incubation with IgGs raised against dissociated RDV suggested that IgGs raised against intact viruses might prevent viral invasion by causing clumping of viruses, thereby reducing the number of infectious units. Our results reveal, for the first time, a possible mechanism for the neutralization, mediated by antibodies, of plant viruses that propagate in insect vector cells.

DOI： 10.6090/jarq.46.65

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

DOI： 10.1128/JVI.00826-11

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC GENERAL MICROBIOLOGY  

The non-structural Pns9 protein of rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. Immunofluorescence and immunoelectron microscopy of RGDV-infected vector cells in monolayers, using antibodies against Pns9 of RGDV and expression of Pns9 in Spodoptera frugiperda cells, demonstrated that Pns9 is the minimal viral factor necessary for formation of viroplasm inclusion during infection by RGDV. When Pns9 in solution was observed under a conventional electron microscope, it appeared as ring-like aggregates of approximately 100 angstrom in diameter. Cryo-electron microscopic analysis of these aggregates revealed cylinders of octameric Pns9, whose dimensions were similar to those observed under the conventional electron microscope. Octamerization of Pns9 in solution was confirmed by the results of size-exclusion chromatography. Among proteins of viruses that belong to the family Reoviridae whose three-dimensional structures are available, a matrix protein of the viroplasm of rotavirus, NSP2, forms similar octamers, an observation that suggests similar roles for Pns9 and NSP2 in morphogenesis in animal-infecting and in plant-infecting reoviruses.

DOI： 10.1099/vir.0.032524-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2011.05.011

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

DOI： 10.1128/JVI.02147-10

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DOI： 10.2210/pdb3vjj/pdb

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その他リンク： http://orcid.org/0000-0001-7071-5093

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2010.03.020

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kappa Da protein from P, furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6 angstrom resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea). (C) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2007.02.075

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記述言語：日本語   出版者・発行元：オプトロニクス社  

CiNii Article

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DOI： 10.5940/jcrsj.58.126

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記述言語：日本語   出版者・発行元：日本放射光学会  

CiNii Article

CiNii Books

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J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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