研究者詳細 - 沈　建仁

Purification and crystallization of oxygen-evolving photosystem II core complex from thermophilic cyanobacteria. 査読国際誌

Jian-Ren Shen, Keisuke Kawakami, Hiroyuki Koike

Methods in molecular biology (Clifton, N.J.) 684 41 - 51 2011年

記述言語：英語掲載種別：研究論文（学術雑誌）

This chapter describes the purification and crystallization of oxygen-evolving photosystem II core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus. Procedures used for purification of photosystem II from the cyanobacterium involves cultivation of cells, isolation of thylakoid membranes, purification of crude and pure photosystem II core complexes by detergent solubilization, followed by differential centrifugation and column chromatography. The purified core dimer particles were successfully used for crystallization, and the methods and conditions used for crystallization are presented. These purification and crystallization procedures can be applied for another thermophilic cyanobacterium T. elongatus.

DOI： 10.1007/978-1-60761-925-3_5

2L1412 光化学系II複合体の原子レベル分解能での結晶構造解析(光生物_光合成,第49回日本生物物理学会年会)

Umena Yasufumi, Kawakami Keisuke, Kamiya Nobuo, Shen Jian-Ren

生物物理 51 S95 2011年

1SL-02 光合成水分解を可能にする光化学系IIの原子構造(1SL 光合成研究で何が明らかにされ、これから何をできるか?～光合成研究の最先端とエネルギー創製研究の現状～,日本生物物理学会第49回年会(2011年度))

沈建仁, Umena Yasufumi, Kawakami Keisuke, Kamiya Nobuo

生物物理 51 S10 2011年

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom, Chaetoceros gracilis 査読国際誌

Ryo Nagao, Akira Moriguchi, Tatsuya Tomo, Ayako Niikura, Saori Nakajima, Takehiro Suzuki, Akinori Okumura, Masako Iwai, Jian-Ren Shen, Masahiko Ikeuchi, Isao Enami

JOURNAL OF BIOLOGICAL CHEMISTRY 285 ( 38 ) 29191 - 29199 2010年9月

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 BIOCHEMISTRY MOLECULAR BIOLOGY INC

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ', PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ', and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl- and Ca2+ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl- and Ca2+ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.

DOI： 10.1074/jbc.M110.146092

Topological Analysis of the Extrinsic PsbO, PsbP and PsbQ Proteins in a Green Algal PSII Complex by Cross-Linking with a Water-Soluble Carbodiimide 査読

Ryo Nagao, Takehiro Suzuki, Akinori Okumura, Ayako Niikura, Masako Iwai, Naoshi Dohmae, Tatsuya Tomo, Jian-Ren Shen, Masahiko Ikeuchi, Isao Enami

PLANT AND CELL PHYSIOLOGY 51 ( 5 ) 718 - 727 2010年5月

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 close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.

DOI： 10.1093/pcp/pcq042

Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium 査読国際誌

Kenji Takasaka, Masako Iwai, Yasufumi Umena, Keisuke Kawakami, Yukari Ohmori, Masahiko Ikeuchi, Yuichiro Takahashi, Nobuo Kamiya, Jian-Ren Shen

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1797 ( 2 ) 278 - 284 2010年2月

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

Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix XI reported previously, in agreement with the recent structure at 2.9 angstrom resolution (A. Guskov, J. Kern. A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 angstrom resolution: role of quinones, lipids, channels and chloride, Nat Struct. Mol. Biol. 16 (2009) 334-342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2009.11.001

Mechanisms of Acido-Tolerance and Characteristics of Photosystems in an Acidophilic and Thermophilic Red Alga, Cyanidium Caldarium 査読

Crystal structure of oxygen evolving Photosystem II at atomic resolution. 査読

Yasufumi Umena, Keisuke Kawakami, Jian-Ren Shen, Nobuo Kamiya

ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 66 S124 - S125 2010年

Enami Isao, Adachi Hideyuki, Shen Jian-Ren

Red Algae in the Genomic Age 13 373 - 389 2010年

記述言語：英語出版者・発行元：Springer

In this chapter, we describe the mechanisms of acido-tolerance in an acidophilic- and thermophilic red alga, Cyanidium caldarium. In spite of the extremely acidic environments it inhabits, the intracellular pH of Cyanidium cells is kept neutral by pumping out the protons previously leaked into the cells according to the steep pH gradient. The H+ pump is driven by the plasma membrane ATPase, utilizing intracellular ATP produced by both oxidative phosphorylation and cyclic photophosphorylation via photosystem I. We also describe the characteristics and function of the two photosystems, Photosystem I (PSI) and II (PSII), in Cyanidium caldarium in comparison with those of cyanobacteria, other eukaryotic algae, and higher plants, based on the crystal structures of the two complexes reported so far.

DOI： 10.1007/978-90-481-3795-4_20

CiNii Article

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography 査読国際誌

Keisuke Kawakami, Yasufumi Umena, Nobuo Kamiya, Jian-Ren Shen

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 21 ) 8567 - 8572 2009年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

The chloride ion, Cl-, is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn4Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl- with a bromide ion (Br-) or an iodide ion (I-) in PSII and analyzed the crystal structures of PSII with Br- and I- substitutions. Substitution of Cl- with Br- did not inhibit oxygen evolution, whereas substitution of Cl- with I- completely inhibited oxygen evolution, indicating the efficient replacement of Cl- by I-. PSII with Br- and I- substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn4Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn4Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl- anions are required to maintain the coordination structure of the Mn4Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.

DOI： 10.1073/pnas.0812797106

Probing tyrosine Z oxidation in Photosystem II core complex isolated from spinach by EPR at liquid helium temperatures. 査読国際誌

Yanan Ren, Chunxi Zhang, Han Bao, Jianren Shen, Jingquan Zhao

Photosynthesis research 99 ( 2 ) 127 - 38 2009年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Tyrosine Z (Tyr(Z)) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr(Z) in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr(Z) oxidation in the S(1) and S(2) states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S(1) state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S(1)Tyr(Z)(*) EPR signal. Furthermore, EPR signals of the S(2) state of the Mn-cluster, the oxidation of the non-heme iron, and the S(1)Tyr(Z)(*) in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.

DOI： 10.1007/s11120-009-9410-x

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga 査読国際誌

Hideyuki Adachi, Yasufumi Umena, Isao Enami, Takahiro Henmi, Nobuo Kamiya, Jian-Ren Shen

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1787 ( 2 ) 121 - 128 2009年2月

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

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms tinder different crystallization conditions. One had a space group of P222(1) with unit cell constants of a = 146.8 angstrom, b = 176.9 angstrom, and c = 353.7 angstrom. and the other one had a space group of P2(1)2(1)2(1) with unit cell constants of a = 209.2 angstrom, b = 237.5 angstrom, and c = 299.8 angstrom. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 angstrom resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 angstrom resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2008.11.004

2P-223 極低温時間分解蛍光測定によるThermosynechococcus vulucanusの光化学系2の二量体と単量体のエネルギー移動の比較(光生物-光合成,第47回日本生物物理学会年会)

Nishi Shunsuke, Komura Masayuki, Noji Tomoyasu, Kawakami Keisuke, Shen Jian-Ren, Shibata Yutaka, Itoh Shigeru

生物物理 49 S142 2009年

3TP2-03 シリカメソ多孔体細孔中に導入した好熱性シアノバクテリア光化学系IIコア複合体の機能(光生物-光合成,第47回日本生物物理学会年会)

Noji Tomoyasu, Kamidaki Chihiro, Kawakami Keisuke, Shen Jian-Ren, Kajino Tsutomu, Fukushima Yoshiaki, Sekitoh Takeshi, Itoh Shigeru

生物物理 49 S60 2009年

Effects of phospholipase and lipase treatments on photosystem II core dimer from a thermophilic cyanobacterium 査読

Jing Leng, Isamu Sakurai, Hajime Wada, Jian-Ren Shen

PHOTOSYNTHESIS RESEARCH 98 ( 1-3 ) 469 - 478 2008年10月

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

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A(2) (PLA(2)) and lipase, and examined their effects on PSII structure and function. PLA(2)-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA(2)- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the Q(B)-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA(2)- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA(2)- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA(2)- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.

DOI： 10.1007/s11120-008-9335-9

Structures and functions of the extrinsic proteins of photosystem II from different species 査読

Isao Enami, Akinori Okumura, Ryo Nagao, Takehiro Suzuki, Masako Iwai, Jian-Ren Shen

PHOTOSYNTHESIS RESEARCH 98 ( 1-3 ) 349 - 363 2008年10月

記述言語：英語出版者・発行元：SPRINGER

This minireview presents a summary of information available on the variety and binding properties of extrinsic proteins that form the oxygen-evolving complex of photosystem II (PSII) of cyanobacteria, red alga, diatom, green alga, euglena, and higher plants. In addition, the structure and function of extrinsic PsbO, PsbV, and PsbU proteins are summarized based on the crystal structure of thermophilic cyanobacterial PSII together with biochemical and genetic studies from various organisms.

DOI： 10.1007/s11120-008-9343-9

Structure and Function of Photosystem II 査読

Jian-Ren Shen, Takahiro Henmi, Nobuo Kamiya

Photosynthetic Protein Complexes: A Structural Approach 83 - 106 2008年9月

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II 査読国際誌

Han Bao, Chunxi Zhang, Keisuke Kawakami, Yanan Ren, Jian-Ren Shen, Jingquan Zhao

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1777 ( 9 ) 1109 - 1115 2008年9月

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

In intact PSII, both the secondary electron donor (Tyrz) and side-path electron donors (Car/Chl(Z)/Cyt(b559)) can be oxidized by P-680(+center dot) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z). EPR signal were independent of the treatment of K3Fe(CN)(6), whereas formation and decay of the Car(+center dot)/Chl(Z)(+center dot) EPR signal correlated with the reduction and recovery of the Fe3+ EPR signal of the non-heme iron in K3Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe3+ reduction, the oxidation of non-heme iron by K3Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe3+ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyrz oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K3Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe3+ state is probably not the intermediate species for the turnover of quinone reduction. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2008.04.044

X-ray crystallographic and biochemical characterizations of a mutant photosystem II complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion mutation 査読国際誌

Takahiro Henmi, Masako Iwai, Masahiko Ikeuchi, Keisuke Kawakami, Jian-Ren Shen, Nobuo Kamiya

JOURNAL OF SYNCHROTRON RADIATION 15 ( Pt 3 ) 304 - 307 2008年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT UNION CRYSTALLOGRAPHY

The crystal structure of a photosystem II (PSII) dimer from Thermosynechococcus vulcanus with its psbTc gene inactivated by insertion mutation of an antibiotic cassette in a site in the C-terminal region was analyzed at 3.8 angstrom resolution. In the crystal structure of the mutant PSII, the transmembrane helix of PsbTc remains, whereas the C-terminal loop of PsbTc has disappeared. In addition, the PsbM subunit, which seemed to be lost in a PsbTc-deletion mutant PSII of T. elongatus, is still present. The deletion of the C-terminal loop of PsbTc in the mutant PSII was verified by mass spectrometry. Thus, the insertion mutation of psbTc eliminated only the C-terminal loop of this subunit. Nevertheless, some features of the mutant PSII, namely a destabilization of the dimeric form and a slight decrease of the oxygen-evolving activity, were observed in the mutant, indicating that the C-terminal loop of PsbTc functions to maintain the stability of the PSII dimer and the activity of oxygen evolution.

DOI： 10.1107/S0909049508002458