SHIRO, Yoshitsugu x (1) 1 2 2 2,3
-40 2MPa (Fe 2 +O 2  ) (IDO) x D (4) CYP105A1 Streptonyces gri sed us la, 25 D3 D3    1α, 25( H 2 D3 X CYP105A1 R84A 25(CHD, D lα   L D- Arg231 (2) 2 19 2,3 8 Å 2 4
1.9 2.4
StaD (3)فBacillus subtillis (H 2 O 2 )
VioBديs 4 (NOR) NOR NO NO Cryo-gas-siter NOR D, IDO Ngb   I <-70 NOR NO FixL ChrS/ChrA cNOR 10 Fab cNOR ABC HmuO hmuO qNOR chrSA qNOR NO qNOR Bacillus stearothermophilus qNOR lmg 75g qNOR 0.1× 0.1× 0.04 ChrS ~3.0 His21 (3)
 2 (7) （ ） ThkA•TrrA ThkA TrrA ThkA•TrrA ATP TrrA HK O-PO 3 2 Image  J RI HK RR ATP N-PO 3 2 ETR1 HK RR FixL/FixJ (1) ETR1 ERS1 Pichia pastoris FixL FixL (2)دو6 1.8 III HK (4) 4 （ ）
NMR NMR Cro (4) NMR s NMR UBL NEDD8 UBL (1) 1 4 Jeyakanthan   4
GFP in vivo in vivo Dronpa GFP BL44B2 Dronpa on-line SS (2)  X X-ray Absorption Spectrum, XAS RCA DNA DNA RCA ssDNA SPring-8 XAS quick-scan (3) DNA RecA DNA DNA RecX recX
NHaseدیNHase (NO) UV/Vis Thiocyanate  Hydrolase (NHase) RecX 54 ssDNA DNA DNA SSB SSB RCA RCA (6) NHase RecX 80 (5) (7) (GDE) GDE GDE GDE 16.5
2 (5)  X
( SS) 4 (WT) oss x 7.8 nm 1SS-1 2SS 1+2 6.7-7.2 nm  3.3-4.9 nm WT SS (6) Gelsolin Villin, adseverin, cap G, advillin, supervillin, flightless I, protovillin, EhABPH X gelsolin
E2 360MPa 12 small heat shock protein (sHSP) SHSP (4) X x 2.1M
native X (OSS)    1 (2SS)  2
(2)
60MPa
X
4.4 nm 9 nm
(1) 0SS s 7 )
7 Metal ions are present in biological system in the form of metal-binding proteins and enzymes, and are involved in physiologically important actions such as biological redox reactions, cellular signal transductions. Research in Bio- metal Science Laboratory focuses on understanding the functions of such metalloproteins and metalloenzymes at molecular and atomic levels on the basis of their molecular structures, which are determined by the SPring-8 RIKEN beam lines.
1. Structural and Functional Analyses of Several Metal-
loenzymes:
(1) Human indoleamine 2,3-dixoygenase (IDO) is a heme-containig dioxygenase and catalyzes the incorporation of dioxygen (O 2 ) into indole rings, that is the first and rate-limiting step in the main pathway of human tryptophan We prepared the 19-type of mutants by catabolism. site-directed mutagenesis and measured their activities of the dioxygenase reaction. The results suggest the important amino acid residues for the catalysis, which helps to under- stand the mechanism of the substrate specificity and the catalytic reaction of IDO. (2) The X-ray diffraction data of CO-bound form of mouse neuroglobin was measured. The difference Fourier maps suggest that upon CO binding the heme group slides into a crevice in the proximal side, which is consistent with the published structure. (3) Peroxygenase P450 is a unique cytochrome P450 type enzyme which catalyzes the hydroxylation reaction of fatty acid using hydrogen peroxide (H 2 O 2 ) as an oxidant. For elucidation of the catalytic mechanism, the crystal structure of the intermediate was determined using x-ray analysis. (4) Cytochrome P450SU-1 (CYP105A1) from Strepto- myces griseolus has an hydroxylation activity for vitamin D 3 to produce the active form of vitamin D 3 , 1 α,25(OH)D 3 . Substrate specificity for vitamin D 3 by P450s is often crucial to their application for analytical and pharmaceutical purpos- es. The crystal structure of P450SU1 in the substrate-free form and the analysis of various mutants and the docking calculation of the CYP105A1-vitamin D 3 provided significant information on the substrate recognition within the catalytic pocket. (5) Staurosporine has attracted much attention due to its powerful anti-tumor activity. StaP and StaD play key roles in staurosporine biosynthesis since main frame of staurosporine is constructed by these enzymes. We have expressed StaP in E. coli and purified by conventional methods. Single crystals of substrate-free and –bound forms which diffract at 1.9- and 2.4-Å resolution were obtained. Crystallographic and bio- chemical analyses of this enzyme are in progress. It was difficult to purify StaD because of its low-heme-content and non-specific degradation, but we managed to obtain high heme-content and pure samples by optimizing expression and purification conditions. We have also started to optimize the conditions of VioB which is a homologue of StaD. (6) Nitric oxide reductase (NOR) is presumed to be an ancestral enzyme of cytochrome c oxidase which is a central enzyme of aerobic respiratory system. In order to obtain short-lived obligatory catalytic intermediate, NOR-NO com- plex, we have developed cryo-gas-citer which diffuses gas molecules into frozen protein solution. Screening buffer condition for cryo-gas-citer experiments reveals lithium chloride, glycerol, or ethylene glycol accelerates diffusion of gas molecules into frozen protein solution. We have been also trying to solve the crystal structure of NOR. We have gener- ated 10 monoclonal antibodies specifically bound to Pseudi- monas aeruginosa cNOR to enhance crystal formation. Using some antibody fragments, crystals of cNOR-Fab complex were obtained. Optimization of the crystallization conditions is in progress. In addition, the crystallization of Bacillus stearothermophilus qNOR, which couples the oxidation of menaquinol with the reduction of NO to N 2 O, was also started. We constructed the expression system of the qNOR in E. coli., and have obtained the single crystals of the purified enzyme which diffracted X-ray at ~3.0Å resolutions. (7) Xray structures of all lipids contained in bovine heart cytochrome c oxidase were determined at 1.8 resolu- tion. The head group of each phospholipid(PL) and the fatty acids of triacylglycerol contributed to the specific bindings. 4PLs stabilized dimmer state and 3PLs in subunit III provided pathway and storage for O 2 .
2. Structural and functional analyses of metalloporteins
relating to cellular signal transduction
The two-component regulatory system is widely distri- It is well known that buted in bacteria, fungi and plants. sensory histidine kinases (HK) sense individual environmen- tal stimuli, and the cognate response regulators (RR) trans- duce their signals downstream upon receiving the phosphoryl group.  However, it has been still unknown how HKs are autophosphorylated upon ligand-binding, and how RRs are activated upon phosphorylation. (1) To elucidate the more details of transduction me- chanism in FixL, the three-dimensional structural information of the full-length FixL is imperative. We have started to try the crystallization of the full-length FixL. As far, we obtained micro crystals which was still insufficient for X-ray diffrac- tion experiments. We will continue the screening of the crystallization conditions to obtain crystals with suitable shapes. (2) Corynebacterium diphtheriae is a causative agent of diphtheria.  Heme oxygenase (HmuO) is involved in iron acquisition from the host heme that has been transported thorough an ABC-type heme transporter.  We indicated that the chrS protein which was produced in the E. coli membrane catalyzed heme-dependent autophosphorylation reaction. Protoporphyrin and its Co and Zn complexes failed in the kinase activation, suggesting that ChrS is a specific sensor for the heme. (3) We constructed the atomic model of ThkA•TrrA complex by the fitting of the domain structures (sensor, dimerization, and catalytic domain of ThkA and TrrA) into the complex structure at the resolution of 4.2 Å.   The struc- ture revealed new insights as follows: [1] the inter-domain beta sheet between sensor and catalytic domain regulates the kinase activity of ThkA, [2] the ATP binding site of catalytic domain changes the conformation after the kinase reaction, and [3] the inter-domain helical interaction between sensor and TrrA regulates the ligand affinity in the sensor domain. We also develop a new detection method for phosphorylated histidine kinase protein using a polyacrylamide gel co-polymerized with the Phos-tag molecule that binds specif- The quantitative a phosphoester dianion (O-PO 3 2 ). ically analysis of the phosphorylated histidine kinase with using of the processing software, Image J, corresponds to the result of a current radioisotope method.  Moreover, this is the first data that Phos-tag binds a phosphoramidate dianion (N-PO 3 2 ). (4) ETR1 and ERS1 are believed to be ethylene receptor proteins containing a cupper ion. However, the transduction mechanism of ethylene concentration remains still unclear. We have tried to construct their overexpression system using yeast ( Pichia pastoris ) as host. Although western-blot analy- sis have not yet indicated the expression of the proteins, use of some variant of Pichia pastoris will be hold promise to establish the construction system of the recombinant proteins.
3. De novo protein design
We had computationally designed a de novo protein that folds into a unique tertiary structure similar to that of a mo- nomeric variant of bacteriophage λ Cro. In the present study, this protein was redesigned to fold cooperatively, based on the solution structure.  The present result indicates that amino acid sequences that realize the folding cooperativity are further selected from those that realize a unique tertiary structure.
4. Development and application of new methods to protein
structural determination
(1) Dronpa is one of switchable fluorescent proteins, which enable us to detect fast movement and tracking of individual proteins in vivo. We introduced lasers and spec- trometer into "on-line" by which we can control the isomeri- zation state of the crystals and subsequently collect X-ray diffraction patters. Based on the structures, we propose a possible mechanism of the reversible photochromism oc- curred in Dronpa (2) We have established the measurement system that suitable for X-ray absorption spectroscopy of metalloproteins and bio-inspired metal complexes in SPring-8 beamlines. A couple of the spectra of structural unknown products of manganese complexes that are stable only in solution at low-temperature were collected by using the system with quick-scan method. The structural information around man- ganese ion was successfully determined by EXAFS analysis. (3) In previous year term, we found evolutionally conserved intermediates between homolog proteins, ubiquitin and NEDD8, which have same topology of ubiquitin-fold (UBL) and are post translational modifiers. This study has been extended to other UBL. Similar intermediates are ob- served in some other post translational modifiers but not in the non-modifiers such as ubiquitin like domains in large proteins. This result suggested the functional relevance of the intermediates to the protein modification systems. Our inter- ests are also focused on the mechanism of amyloid fibril formation in proteins. The disulfide-deficient variant of hen lysozyme was used as a model amyloid protein. The kinetic mechanism of dissociation of the protofibril was studied in detail by pressure-jump Trp fluorescence spectroscopy. (4) RecA protein plays a central role in homologous recombination by promoting DNA strand exchange.  Al- though RecX protein downregulates the RecA function, the mechanism is not clear.   Therefore, we prepared T. thermo- philus RecX protein and examined its properties. RecX protein existed as a monomer, showed 54% α-helical content and was stable up to 80 °C. In addition, RecX protein interacted with T. thermophilus RecA protein directly and inhibited the ssDNA-dependent ATPase and strand exchange activities in vitro. (5) Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become popular in the amplifica- tion of template DNA.  We constructed some mutant SSB proteins from T.thermophilus to increases the efficiency of DNA synthesis and the accuracy of RCA.   As a result, we succeeded to get one of the SSB mutant protein, which halved the elongation time required to synthesize DNA and elimi- nated nonspecific DNA products in RCA reactions. (6) To gain a deeper understanding of the reaction mechanism, we are developing techniques of time-resolved crystallography. Nitrile Hydratase (NHase) from Rhodococ- cus is an enzyme capable of catalyzing hydration of nitriles, and it contains a mononuclear non-heme iron as its reaction center. The iron center is activated by photo-driven NO release. This year we refined structures obtained last year, and verification and obtained supporting data by UV/Vis mi- cro-spectro photometer. And, determination of Thiocyanate Hydrolase, that is a new nitrile hydratase family protein, structure by X-ray crystallography. (7) Glycerophosphodiester phosphodiesterase (GDE) catalyzes the hydrolysis of glycerophosphodiesers, degrada- products of phospholipids, producing glycerol tion 3-phosphate and corresponding alcohols. Among mammalian GDEs, only GDE1 is reported to have GDE activity. We have constructed an expression system for 6 kinds of mouse GDEs. GDEs are expressed in insect cells and the purified protein was obtained. We have analyzed the enzyme activity of a mouse GDE. Screening of crystallization was successful in several conditions. 5. Structural studies on protein solutions using synchro-
tron small-angle X-ray scattering
RIKEN structural beamline I, BL45XU-SAXS is ac- tively used by Fujisawa and his collaborators including inside and outside RIKEN.  The main focus of his studies is the kinetic studies of protein in solution that include submillise- cond fast-flow measurements and high pressure experiments. The high pressure apparatus developed by his group has a very small sample volume and can record time-resolved data with transient change of pressures.  The pressure perturba- tion was used for studying weak interaction between proteins, subunits and domains. The physical properties of filamen- taneous proteins, high molecular protein complex, and intact crystalline were studied.
6. Supporting system for structural biology research in
Harima Institute
We supported researchers of the Harima Institute in protein characterization.   This year, more than 1200 protein samples have been characterized by protein sequencer, mass spectrometer, PAGE, HPLC, fluorescence spectrometer and others.
Staff
Head Dr. Yoshitsugu SHIRO
Members
Dr. Hiroshi AOYAMA Dr. Tetsuro FUJISAWA Dr. Yasuhiro ISOGAI Dr. Yoshiaki KAWANO Dr. Akihiro KIKUCHI Dr. Tsutomu MIKAWA Dr. Shingo NAGANO Dr. Hiro NAKAMURA Dr. Hiroshi SUGIMOTO Dr. Tomoya HINO  ¹  ² Dr. Misa KIM Dr. Noriyasu OHSHIMA   ¹ Dr. Seiji YAMADA  ¹ Dr. Satoshi HIRANO  ² Dr. Jeyaraman JEYAKANTHAN  ² Dr. Atsuko KOHARA  ² Dr. Yushi MATSUMOTO  ² Dr. Mitsuhiro NAKAMURA  ² Mr. Jun-ichiro TAKA   ² Dr. Eduardo VOTTERO  ² Mr. Hiroshi MATSUO  ³ Ms. Naoko TAKAHASHI  ³ 1 Special Postdoctral Researcher ² Contract Researcher 3 Contract Technical Scientist Visiting Members Dr. Shin-ichi ADACHI (PF, KEK) Prof. Kazuyuki AKASAKA (Sch. of Biol.-Oriented Sci. and Technol., Kinki Univ.) Dr. Yoshio GOTO (Sony Corporation) Prof. Yoshiki HIGUCHI (Grad. Sch. of Life Sci., Univ. of Hyogo) Prof. Masao IKEDA-SAITO (IMRAM, Tohoku Univ.) Mr. Manabu ISHIDA (Res. Center for Micro-Nano Technol., Hosei Univ.) Prof. Yutaka ITOH (Tokyo Metropolitan Univ.) Dr. Takashi IYANAGI Dr. Hirofumi KOMORI (Grad. Sch. of Life Sci., Univ. of Hyogo) Prof. Tsutomu KOYAMA (Grad. Sch. Sci., Nagoya Univ.) Prof. Yukio MORIMOTO (Res. Reactor Inst., Kyoto Univ.) Prof. Takumi NOGUCHI (Inst. Mater. Sci., Univ. Tsukuba) Dr. Hitomi SAWAI (Okazaki Inst. for Integrative Bioscience, National Inst. of Natural Sci.) Dr. Naoki SHIBATA (Grad. Sch. of Life Sci., Univ. of Hyogo) Dr. Yasuhito SHOMURA (Grad. Sch. of Life Sci., Univ. of Hyogo) Prof. Chikashi TOYOSHIMA (IMCB, Univ. of Tokyo) Dr. Masaki UNNO (IMRAM, Tohoku Univ.) Mr. Mamoru YAMANISHI (Okayama Univ.) Prof. Shigeyuki YOKOYAMA (Sch. of Sci., Univ. of Tokyo) Prof. Shin-ya YOSHIKAWA (Grad. Sch. of Life Sci., Univ. of Hyogo)
Trainees
Ms. Eiko FUKUMURA (Grad. Sch. of Sci., Osaka Univ.) Mr. Kazumi HATA (Grad. Sch. of Biol.-Oriented Sci. and Technol., Kinki Univ.) Mr. Hiroshi IMAMURA (Ritsumeikan Univ. Grad. Sch.) Ms. Miki KOBAYASHI (Grad. Sch. of Life Sci., Univ. of Hyogo) Ms. Erisa KOMETANI (Grad. Sch. of Life Sci., Univ. of Hyogo) Mr. Shusuke KUSAMA (IMRAM, Tohoku Univ.) Mr. Akihiro MAENO (Grad. Sch. of Biol.-Oriented Sci. and Technol., Kinki Univ.) Mr. Masatomo MAKINO (Grad. Sch. of Life Sci., Univ. of Hyogo) Mr. Masakazu MORIYAMA (Grad. Sch. of Life Sci., Univ. of Hyogo) Mr. Keita YAMAMOTO (Grad. Sch. of Life Sci., Univ. of Hyogo) Mr. Norihiko YOKOI (Grad. Sch. Sci., Nagoya Univ.) Ms. Keiko YOTSUYA (Grad. Sch. of Life Sci., Univ. of Hyogo) [
]
Publ i cat i ons
Hirotsu S., Chu G. C., Unno M., Lee D.-S., Yoshida T., Park S.-Y., Shiro Y., and Ikeda-Saito M. : "The Crystal Struc- tures of the Ferric and Ferrous Forms of the Heme Com- plex of HmuO, a Heme Oxygenase of Corynebacterium diphtheriae", J. Biol. Chem. 279, 11937-11947 (2004). Takeda K., Matsui Y., Kamiya N., Adachi S., Okumura H., and Kohyama T. : "Crystal structure of the M Intermediate of Bacteriorhodopsin: allosteric structural changes me- diated by sliding movement of a transmembrane helix", J. Mol. Biol. 341, 1023-1037 (2004). Makino M., Sugimoto H., Sawai H., Kawada N., Yoshizato K., and Shiro Y. : "High-resolution structure of human cytoglobin: identification of extra N- and C-termini and a new dimerization mode", Acta Cryst. D 62 , 671-677 (2006). Nakano N., Okazaki N., Sato S., Takio K., Kuramitsu S., Shinkai A., and Yokoyama S. : "Structure of the stand-alone RAM-domain protein from Thermus thermo- philus HB8", Acta Cryst. F 62, 855-860 (2006). Nishimiya Y., Kondo H., Yasui M., Sugimoto H., Noro N., Sato R., Suzuki M., Miura A., and Tsuda S. : "Crystalliza- tion and preliminary X-ray crystallographic analysis of Ca 2+ -independent and Ca 2+ -dependent species of the type II antifreeze protein", Acta Cryst. F 62 , 538-541 (2006). Isogai Y. : "Native protein sequences are designed to desta- bilize folding intermediates", Biochemistry 45 , 2488-2492 (2006). Tanaka A., Nakamura H., Shiro Y., and Fujii H. : "Roles of the heme distal residues of FixL in O 2 sensing: A single convergent structure of the heme moiety is relevant to the downregulation of kinase activitiy", Biochemistry 45 , 2515-2523 (2006). Makino A., Ishii K., Murate M., Hayakawa T., Suzuki Y., Suzuki M., Ito K., Fujisawa T., Matsuo H., Ishitsuka R., Kobayashi T. and "D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-pro panol  alters cellular cholesterol homeostasis by mod- ulating the endosome lipid domains", Biochemistry 45 , 4530-4541 (2006). Hayakawa T., Hirano Y., Makino A., Michaud S., Lagarde M., Pageaux J. F., Doutheau A., Ito K., Fujisawa T., Ta- kahashi H., and Kobayashi T. : "Differential Membrane of Packing of Stereoisomers 45, Bis(monoacylglycero)phosphate", Biochemistry 9198-9209 (2006). Yoshikawa S., Muramoto K., Shinzawa-Itoh K., Aoyama H., Tsukihara T., Ogura T., Shimokata K., Katayama Y., and Shimada H. : "Reaction mechanism of bovine heart cy- tochrome c oxidase", Biochim. Biophys. Acta 1757, 395-400 (2006). Yoshikawa S., Muramoto K., Shinzawa-Itoh K., Aoyama H., Tsukihara T., Shimokata K., Katayama Y., and Shimada H. : "Proton pumping mechanism of bovine heart cytoch- rome c oxidase", Biochim. Biophys. Acta 1757, 1110-1116 (2006). Takarada H., Kawano Y., Hashimoto K., Nakayama H., Ueda S., Yohda M., Kamiya N., Dohmae N., Maeda M., and Odaka M. : "Mutational study on αGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771", Biosci. Biotechnol. Biochem. 70 , 881-889 (2006). Kumar P. K., Thirumananseri K., and Mizuno H. : "Structur- al basis of HutP-mediated transcription anti-termination", Curr. Opin. Structural Biol. 16 , 18-26 (2006). Hayashi T., Murata D., Makino M., Sugimoto H., Matsuo T., Sato H., Shiro Y., and Hisaeda Y. : "Crystal Structure and peroxidase activity of Myoglobin reconstituted with iron porphycene", Inorg. Chem. 45 , 10530-10536 (2006). Uzawa T., Kimura T., Ishimori K., Morishima I., Matsui T., Saito M. I., Takahashi S., Akiyama S., and Fujisawa T. : "Time-resolved Small-Angle X-ray Scattering Investiga- tion on the Folding Dynamics of Heme Oxygenase: Im- plication of the Scaling Relationship for the Submillise- cond Intermediates of Protein Folding", J. Mol. Biol. 357, 997-1008 (2006). Park S.-Y., Yokoyama T., Shibayama N., Shiro Y., and Tame J. : "1.25 Å resolution crystal structures of human Haemoglobin in the Oxy, Deoxy and Carbonmonoxy Forms", J. Mol. Biol. 360, 690-701 (2006). Inagaki E., Ohshima N., Takahashi H., Kuroishi C., Yo- koyama S., and Tahirov T. : "Crystal structure of Thermus thermophilus δ1-Pyrroline-5-carboxylate dehydrogenase", J. Mol. Biol. 362 , 490-501 (2006). Yamada S., Akiyama S., Sugimoto H., Kumita H., Ito K., Fujisawa T., Nakamura H., and Shiro Y. : "The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering", J. Mol. Biol. 362, 123-139 (2006). Kitahara R., Yamaguchi Y., Sakata E., Kasuya T., Tanaka K., Kato K., Yokoyama S., and Akasaka K. : "Evolutionally conserved intermediates between ubiquitin and NEDD8", J. Mol. Biol. 363, 395-404 (2006). Eleonora S. V., Vladimir V. V., Huajie W., Fujisawa T., and Jin W. Y. : "Autoxidized Phospholipids in Hexane: Na- no-self-assemblies Studied by Synchrotron Small-Angle X-ray Scattering", Langmuir 22, 7994-8000 (2006). Kitahara R., Okuno A., Kato M., Taniguchi Y., Yokoyama S., and Akasaka K. : "Cold denaturation of ubiquitin at high pressure", Magn. Reson. Chem. 44, S108-S113 (2006). Kashiwagi K., Isogai Y., Nishiguchi K., and Shiba K. : "Frame shuffling: a novel method for in vitro protein evo- lution", Protein Eng. 19, 135-140 (2006). Okazaki N., Kumei M., Manzoku M., Kuramitsu S., Shirou- zu M., Shinkai A., and Yokoyama S. : "Structure of a UPF0150-family protein from Thermus thermophilus HB8", Acta Cryst. F 63 , 173-177 (2007). Kanaujia S. P., Ranjani C. V., Jeyaraman J., Baba S., Chen L., Liu Z., Wang B., Nishida M., Ebihara A., Shinkai A., Kuramitsu S., Shiro Y., Sekar K., and Yokoyama S. : "Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus " , Acta Cryst. F 63 , 27-29 (2007). Kanaujia S. P., Ranjani C. V., Jeyaraman J., Baba S., Kuroi- shi C., Ebihara A., Shinkai A., Kuramitsu S., Shiro Y., Sekar K., and Yokoyama S. : "Cloning, expression, puri- fication, crystallization and preliminary X-ray crystallo- graphic study of DHNA synthetase from Geobacillus kaustophilus", Acta Cryst. F 63, 103-105 (2007). Yamada S., Nakamura H., Kinoshita E., Kinoshita-Kikuta E., Koike T., and Shiro Y. : "Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis", Anal. Biochem. 360 , 160-162 (2007). Shibata R., Bessho Y., Shinkai A., Nishimoto M., Fusatomi E., Terada T., Shirouzu M., and Yokoyama S. : "Crystal structure and RNA-binding analysis of the archaeal tran- scription factor NusA", Biochem. Biophys. Res. Commun.
355, 122-128 (2007).
Lokanath N. K., Matsuura Y., Kuroishi C., Takahashi N., and Kunishima N. : "Dimeric core structure of modular stator subunit E of archaeal H + -ATPase", J. Mol. Biol. 366 , 933-944 (2007). Kohara A., Nakajima C., Yoshida S., and Muranaka T. : "Characterization and engineering of glycosyltransferases responsible for steroid saponin biosynthesis in Solana- ceous plants", Phytochemistry 68 , 478-486 (2007).
Oral Presentations
( ) Fujisawa T. : "The use of the third generation synchrotron small-angle scattering on protein dynamics in solution: present and future", 5th East Asian Biophysics Sympo- sium and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2004). Nagano S. and Poulos T. L. : "Crystal structures of ferrous oxygen-complex of wild-type cytochrome P450cam and its mutants, D251N and T252A: implication for oxygen activation by P450", 14th Takeda Science Foundation Symposium on Bioscience: The 50th Anniversary of Oxygenases Advances and Reflections, Kyoto, Apr. (2006). Shiro Y. : "Structural basis of O 2 incorporation into Indole Ring Catalyzed by Indoleamine 2,3-Dioxygenase", 14th Takeda Science Foundation Symposium on Bioscience: The 50th Anniversary of Oxygenases Advances and Ref- lections, Kyoto, Apr. (2006). Fujisawa T. : "Interpretation of SAXS Data", Innovation in Nanoscale Modeling and Imaging of Biological System (Innovations 2006), Huston, USA, Apr. (2006). Shiro Y. : "Redox chemistry of NO and O ⁄  in hemoproteins", Post Hayaishi Symposium at SPring-8 "Chemical Biology of Redox Metalloenzymes", Harima, Apr. (2006). Fujita M., Sawayanagi T., Tanaka T., Iwata T., Abe H., Doi Y., Ito K., and Fujisawa T. : "Cynchrotron SAXS and WAXD studies on sructural change of Poly[(R)-3-hydroxybutyrate] single crystals on heating", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, June (2006). Iyanagi T., Yamamoto K., and Shiro Y. : "CaM activates one-electron reduction of quinones by neuronal nitric oxide synthase", 20th IUBMB Int. Congress of Biochemi- stry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Sugimoto H., Shinkyo R., Yoneda S., Kamakura M., Ikushi- ro S., Shiro Y., and Sakaki T. : "Crystal structure of cy- tochrome P450SU-1 (CYP105A1) from Streptomyces griseolus and molecular docking with its substrate vita- mine D 3 ", 20th IUBMB Int. Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Hino T., Kumita H., Fukumori Y., and Shiro Y. : "Crystallo- graphic study of bacterial nitric oxide reductase from Pseudomonas aeruginosa", 20th IUBMB Int. Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Fukumura E., Sugimoto H., Nagano S., Takio K., Iyanagi T., and Shiro Y. : "L-Tryptophan 2,3-dioxygenase: expression, purification of recombinant enzyme and characterization of its enzymatic properties", 20th IUBMB Int. Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Shiro Y. and Sugimoto H. : "Reaction mechanism of heme-containing oxygenases based on their molecular structures", 20th IUBMB Int. Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Yamada S., Akiyama S., Sugimoto H., Kumita H., Ito K., Fujisawa T., Nakamura H., and Shiro Y. : "Signaling pathway and regulatory mechanism of sensor histidine ki- nase in two-component system", 20th IUBMB Int. Con- gress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Shinkyo R., Sugimoto H., Kamakura M., Ikushiro S., Shiro Y., and Sakaki T. : "Structure-function analysis of Strep- tomyces griseolus CYP105A1 catalyzing hydroxylation of vitamin D 3 ", 20th IUBMB Int. Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, June (2006). Takahashi S. and Fujisawa T. : "Collapse and Search" Dy- namics of Protein Folding Detected by Time-resolved Small Sngle X-ray Scattering", 2nd Int. Sympo. on the Frontier of Applied Mathematics, (Tsinghua University), Beijing, China, June (2006). Aoyama H. : "Towards X-ray structural determination of hydrogen atoms in bovine heart cytochrome c oxidase", Workshop on Reaction Mechanisms of Ener- Int. gy-Transducing Metalloenzymes, Harima, June (2006). Sugimoto H. : "X-ray structural studies on Indoleamine Dioxygenase", Int. Workshop on Reaction Mechanisms of Metalloenzymes, Energy-Transducing June Harima, (2006). Yamada S., Nakamura H., and Shiro Y. : "Inter-domain and inter-molecular signaling pathway in two component sys- tem: Histidine kinase and response regulator", Satellite Meeting of the 20th IUBMB Int. Congress of Biochemi- stry and Molecular Biology and 11th FAOBMB Congress, Sendai, June (2006). Sugimoto H. : "Structural biology of a Tryptophan-Ring cleavage Enzyme : X-ray structure and reaction mechan- ism of human indoleamine 2,3-dioxygenase", 11th Meet- ing of Int. Study Group for Tryptophan Research (IS- TRY-2006 Tokyo), Tokyo, July (2006). Matsufuji T., Jinbo Y., Izumi Y., Kumano H., and Fujisawa T. : "Characterization of transient intermediates in the dissociation kinetics of calcium-calmodulin-peptide com- plex", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Iwamoto H., Inoue K., Fujisawa T., and Yagi N. : "Evolution of insect flight muscle structure as viewed from x-ray fiber diffraction", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Toba S., Iwamoto H., Fujisawa T., Sakakibara H., and Oiwa K. : "Fiber diffraction study on flagellar axonemes of chlamydomonas and the small angle x-ray scattering analysis on the inner-arm dynein c", 13th Int. Conf. on Small-angle Scattering (SAS2006)    , Kyoto, July (2006). Akiyama S., Yamada S., Sugimoto H., Kumita H., Ito K., Fujisawa T., Nakamura H., and Shiro Y. : "Signal trans- duction pathway in Histidine Kinase and Response Regu- lator Complex revealed by joint usage of Crystallography and small-angle X-ray scattering", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Takahashi H., Hayakawa T., Kawasaki Y., Ito K., Fujisawa T., Kodama M., and Kobayashi T. : "Structural characte- rization of N-Lignoceroyl(C24:0) Sphingomyelin bilayer membranes by SAXS and XRD : a reevaluation", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Fujisawa T., Kuwamoto S., Maeda Y., and Okamoto Y. : "The mechanism of pressure-induced oligomerization of heavy meromyosin molecules probed by synchrotron small-angle X-ray scattering", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Kamimura S., Tamura T., Wakayama J., Noda N., Sugiyama T., Fujisawa T., and Iwamoto H. : "X-ray diffraction analysis of the axonemal structure of sea-urchin sperm flagella", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Takahashi S. and Fujisawa T. : "Collapse and search" dy- namics of protein folding detected by time-resolved small angle X-ray scattering", 13th Int. Conf. on Small-angle Scattering (SAS2006), Kyoto, July (2006). Jeyaraman J., Kuroishi C., Kuramitsu S., Yokoyama S., and Shiro Y. : "Crystal Structure of Corbonic anhydrase Com- plexed with Bicarbonate from Pyrococcus horikoshii OT3", 2006 Meeting of the American Crystallographic Association (ACA 2006), Honolulu, USA, July (2006). Kitahara R., Yokoyama S., and Akasaka K. : "A new para- digm of protein structures by variable pressure NMR", IMS Symposium on New Frontiers of NMR Molecular Science, Okazaki, July (2006). Hatanaka M., Honda M., Ishibe S., Mikawa T., Ito Y., Shi- bata T., and Yamazaki T. : "Observation of structural changes induced by ATP hydrolysis in a microcrystalline sample of protein-DNA filaments by Solid-State NMR", 22nd Int. Conf. on Magnetic Resonance in Biological Systems (ICMRBS 2006), Gottingen, Germany, Aug. (2006). Maeno A., Kitahara R., Dahlquist F., Yokoyama S., Mulder F., and Akasaka K. : "Variable pressure NMR reveals conformational fluctuation around cavities in a protein", 22nd Int.  Conf. on Magnetic Resonance in Biological Systems (ICMRBS 2006), Gottingen, Germany, Aug. (2006). Kitahara R., Hata K., Yokoyama S., Akasaka K., and Cheng J. : "Variable pressure NMR reveals the energy landscape of BTK SH3 domain", 22nd Int. Conf. on Magnetic Re- sonance in Biological Systems (ICMRBS 2006), Gottin- gen, Germany, Aug. (2006). Simorellis A., Kitahara R., Yokoyama S., Akasaka K., and Koide S. : "Variable pressure NMR study on multiple conformational states of OspA, a vaccine candidate for Lyme disease", 22nd Int.   Conf. on Magnetic Resonance in Biological Systems (ICMRBS 2006), Gottingen, Ger- many, Aug. (2006). Kitahara R., Yokoyama S., Akasaka K., and Kato K. : "Va- riable pressure NMR reveals similarities and differences in structure and pro- dynamics of ubiquitin-like teins/domains", Discussions on Protein Hydration and Structural Dynamic: High Pressure and other approaches, Montpellier, France, Aug. (2006). Kitahara R., Simorellis A., Maeno A., Yokoyama S., Koide S., and Akasaka K. : "Variable pressure NMR and fluo- rescence studies on multiple conformational states of Os- pA, a vaccine candidate for Lyme disease", 4th Int. Conf. High Pressure on Bioscience and Biotechnolo- gy(HPBB2006), Tsukuba, Sept. (2006). Aoyama H. : "Structures and functions of lipids in bovine heart cytochrome c oxidase", SLS Seminar, Zurich, Swit- zerland, Sept. (2006). Kitahara R., Yokoyama S., Akasaka K., and Kato K. : "Si- milarities and differences in structure and dynamics of proteins/domains", Switzerland-Japan ubiquitin-like Sympo. on Structural Biology 2006, Brunnen, Switzerland, Sept. (2006). Fujisawa T. : "Overview of biological small-angle scattering at SPring-8", 1st EMBL Beamline Workshop `BIOSAXS @ PETRA-III', (European Molecular Biology Laboratory), Hamburg, Germany, Oct. (2006). Fujisawa T. : "Time-resolved X-ray scattering", EMBO Practical Course on Solution Scattering from Biological Macromolecule (European Molecular Biology Labora- tory), Hamburg, Germany, Oct. (2006). Takai Y., Ota M., and Isogai Y. : "A method to evaluate heme-binding affinity on the protein theoretical model", 5th East Asian Biophysics Symposium and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Ishida M., Yasuda A., Murakami Y., Ota M., Isogai Y., and Imai K. : "Ancestral myoglobins: amino acid sequences, syntheses and oxygen-binding properites", 5th East Asian Biophysics Sympo.   and 44th Annual Meeting of the Bi- ophysical Society of Japan (EABS and BSJ 2006), Oki- nawa, Nov. (2006). Yamada S., Sugimoto H., Kobayashi M., Akiyama S., Na- kamura H., and Shiro Y. : "Crystal structure of histidine kinase and response regulator complex", 5th East Asian Biophysics Sympo.   and 44th Annual Meeting of the Bi- ophysical Society of Japan (EABS and BSJ 2006), Oki- nawa, Nov. (2006). Hino T., Kumita H., Fukumori Y., and Shiro Y. : "Crystalli- zation of bacterial nitric oxide reductase using monoclonal antibody fragment", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Imamura H., Isogai Y., and Kato M. : "Difference of folding properties between natural and de novo cro proteins", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Toba S., Iwamoto H., Fujisawa T., Sakakibara H., and Oiwa K. : "Fiber diffraction study on flagellar axonemes from a wild type and mutant strains of chlamydomonas", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Lokanath N. K., Nakamura M., Ohshima N., Takio K., and Kunishima N.    : "First crystal structure of the 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Uzawa T., Kimura T., Ishimori K., Morishima I., Matsui T., Ikeda-Saito M., Takahashi S., Akiyama S., and Fujisawa T. : "Generality of initial collapse demonstrated by scaling relationship for submillisecond intermediates of protein folding", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Aoyama H., Shinzawa-Itoh K., Maeda T., Tsukihara T., and Yoshikawa S. : "Lipid movements in bovine heart cy- tochrome c oxidase depending on crystal packing", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Iwamoto H., Inoue K., Fujisawa T., and Yagi N. : "Molecu- lar mechanism of stretch activation in insect flight muscle as examined by X-ray diffraction", 5th East Asian Bio- physics Sympo. and 44th Annual Meeting of the Biophys- ical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Kitahara R., Yokoyama S., Akasaka K., and Kato K. : "Probing energy landscape of ubiquitin-like proteins with variable pressure NMR", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical So- ciety of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Sugimoto H. and Shiro Y. : "Structural basis of O 2 incorpo- ration into indole-ring catalyzed by human indoleamine 2,3-dioxygenase", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Takahashi H., Hayakawa T., Kawasaki Y., Ito K., Fujisawa T., Kodama M., and Kobayashi T. : "Structural investiga- tion on highly asymmetric sphingomyelin (C24:0 SM) bi- layers", 5th East Asian Biophysics Sympo and 44th An- nual Meeting of the Biophysical Society of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Isogai Y. : "Structure-based design of folding cooperativity of a de novo cro protein", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical So- ciety of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Kamimura S., Iwamoto H., and Fujisawa T. : "X-ray diffrac- tion from dynein motors and microtubles in sea-urchin sperm flagellar axonemes", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical So- ciety of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Aoyama H., Shinzawa-Itoh K., Tsukihara T., and Yoshikawa S. : "X-ray structural determination of hydrogen atoms in bovine heart cytochrome c oxidase", 5th East Asian Bio- physics Sympo. and 44th Annual Meeting of the Biophys- ical Society of Japan (EABS and BSJ 2006)   Okinawa Nov. (2006). Takahashi S. and Fujisawa T. : "Collapse and search" dy- namics of protein folding detected by time-resolved small-angle x-ray scattering", 5th East Asian Biophysics Sympo. and 44th Annual Meeting of the Biophysical So- ciety of Japan (EABS and BSJ 2006), Okinawa, Nov. (2006). Shibata R., Bessho Y., Shinkai A., Nishimoto M., Fusatomi E., Terada T., Shirouzu M., and Yokoyama S. : "Crystal structure of the archaeal transcription termination factor NusA", Joint Conf. of the Asian Crystallographic Associa- tion and the Crystallographic Society of Japan (As- CA'06/CrSJ), Tsukuba, Nov. (2006). Shiro Y. : "Redox Chemistry of Hemoproteins", Kyoto Conf. on Solid State Chemistry Transition Metal Oxides: Past, Present and Future, Kyoto, Nov. (2006). Fujisawa T. and Takahashi S. : "Protein folding dynamics detected by time-resolved synchrotron x-ray small-angle scattreing technique", 2nd Int. Sympo. on Portable Syn- chrotron Light Sources and Advanced Applications, (Rit- sumeikan University), Kusatsu, Shiga Pref. Jan. (2007). Kitahara R. : "Pressure and Protein Dynamics", 3rd Japa- nese-french Seminar on Structural Dynamics of Proteins, Grenoble, France, Jan. (2007). Kitahara R., Gallopin M., Yokoyama S., Guittet E., Rou- mestand C,    Heijenoort C.V., and Akasaka K. : "Pressure NMR for protein dynamics", 20eme congres du GERM 2007, Perpignan, France, Mar. (2007). Toba S., Iwamoto H., Fujisawa T., Sakakibara H., and Oiwa K. : "Conformational changes of flagellar axonemes re- vealed by fiber diffraction study of flagella from chlamy- domonas strains", 51st Annual Meeting of Biophysical Society, (Biophysical Society), Boltimore, USA, Mar. (2007). Shiro Y. : "Intra-and Intermolecular signal transduction of Two-Component regulatory system", Int. Sympo. on Bio- logical Application of Vibrational Spectroscopy, Harima, Mar. (2007). , : "OS TIHA0829 5 8 (2006). : "Therms thernophilus HB8 cANP ". 8 (2006). , NisA X ", / 10 ( 2006 ). : "NR Phot oconversi on 1 : " ", : " X ", / 1  11 (2006). : "SPring-8 . (SPring-8), , Callopin M, Rounestand C, , Quittet E , conbinatry use of relaxation dispersion and variable pressure NR for protein dynamics analysis: D  donain of  annexin I",      45   NR 11 ( 2006). , . , . 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