シンクロトロン放射による細胞内システムの動的構造解析

1991 Fiscal Year Annual Research Report

• Project/Area Number: 02302092
• Research Institution: Osaka University
• Principal Investigator: 徳永 史生 大阪大学, 理学部, 教授 (80025452)
• Co-Investigator(Kenkyū-buntansha): 三井 利夫 明治大学, 理工学部, 教授 (70000726) ; 江橋 節郎 岡崎国立共同研究機構, 機構長 (10009863) ; 池上 明 慶応義塾大学, 医学部, 教授 (60011483) ; 飯塚 哲太郎 理化学研究所, 主任研究員 (30029475) ; 安藤 正海 高エネルギー物理学研究所, 放射光実験施設, 教授 (30013501)
• Keywords: シンクロトロン放射 / 細胞内システム / 動的構造 / 蛋白質 / 結晶解析 / 筋肉 / 生体膜 / 生物物理

Research Abstract

最近シンクロトロン放射中の強力X線を利用した回析・散乱実験が急速に進展し、結晶中での酵素反応を高時間分解能で解析する研究が始まり、また筋収縮の分子的機構や膜蛋白機能の解明への動的構造解析が出来るようになった。本総合研究はシンクロトロン放射の利用者を中心に種々の蛋白質や生体中の超分子構造の動的構造解析について現在の問題点を明らかにし、この分野の研究を促進することを目的とした。本年度は巨大分子用ワイセンベルグカメラとイメイジングプレ-トを持つ巨大分子結晶構造解析システムを開発しAIDSウイルスが増殖するときに必要な逆転写酵素の結晶を解析し、性能が世界の他の設備より優っていることを確認した。また全自動迅速X線回折計を開発し、テトラゾ-ル・ミオグロビンや茸レクチンで性能評価した。 さらにゾ-ンプレ-トを用いた結像型軟X線顕微鏡を開発し、赤血球などの生体物質を観察しその有効性を確認した。 蛋白質の結晶解析では、免疫グロブリンのFvフラグメント・古細菌フェレドキシン・硫酸還元菌のチトクロムC_3、C_<553>・ヒドロゲナ-ゼ・高分子チトクロムなどの構造を解析した。X線吸収スペクトル法で、酸化型チトクロムP450と過酸化酵化酵素やミオグロビンと比較したところ、鉄と配位子との結合の性質の差異が分かった。時分割X線小角散乱によりATP合成酵素のα^3β^3複合体がATPを加水分解してαβに解離する過程を追跡し、反応過程を解析した。また紫膜は光反応中に結晶構造の乱れが起こらないことが分かった。癌遺伝子産物rasp21のGTP・GDP交換反応に伴う構造変化を変異体も含めて解析し、その機能発現の機構を考察した。 筋肉については収縮中の細いフィラメント構造変化及びアクチン・ミオンシン系の微細構造変化を解析した。またミオシン頭部の化学修飾の影響を調べた。光照射に伴うイカの視細胞微絨毛の構造変化を解析し、 昨年よりより精密なモデルが出来た。

Research Products

• [Publications] Akiba,T.: "Monolayer Crystailization of flagellar LーP Rings by Sequential Addition and Depletion of Lipid" Science. 252. 1544-1546 (1991)
• [Publications] Amemiya,Y.: "Imaging Plate and its application To Xーray diffraction of muscle" Adv.Biophys.27. 115-127 (1991)
• [Publications] Ebashi,S.: "Excitationーcontraction couplig and the mechanism of muscle contraction" Annu.Rev.Physiol.53. 1-16 (1991)
• [Publications] Ebashi,S.: "Muscle research:A cornerstone of life science" Med.Sport Sci.37. 1-6 (1991)
• [Publications] Flanagan,J.M.: "Truncated Staphylococcal Nuclease is Compact but Disordered" Proc.Natl.Acad.Sci.U.S.A.89. 748-752 (1992)
• [Publications] Fujii,I.: "Evaluation of xーray Diffraction Data from Protein Crystals by Use of an Imaging Plate" Acta.Cryst.B47. 137-144 (1991)
• [Publications] 井上 英男: "放射光を用いた生体高分子の構造研究ー小角X線散乱測定による生体反応の研究ー" 島津評論. 48. 265-276 (1991)
• [Publications] 片岡 幹雄: "カルモジュリンの溶液構造" 生物物理. 31. 293-298 (1991)
• [Publications] 片岡 幹雄: "蛋白質の溶液構造と動力学" 日本物理学会誌. 46. 1040-1042 (1991)
• [Publications] 佐藤 文俊: "蛍光XAFSの実験技術その1:金属蛋白質の蛍光X線強度と測定装置の配置との関係" 放射光. 4. 151-156 (1991)
• [Publications] 佐藤 能雅: "シクロトロン放射光と蛋白質結晶解析" 蛋白質核酸酵素. 36. 332-344 (1991)
• [Publications] 佐藤 能雅: "タンパク質の活性、機能と進化の情報を与える立体構造" 現代化学. 245. 24-31 (1991)
• [Publications] 徳永 史生: "異常条件下の生物ー高度好塩菌と紫膜" 化学と工業. 44. 1113-1117 (1991)
• [Publications] 江橋 節郎: "医科学を語るー研究する楽しみ" 実験科学. 9. 1778-1786 (1991)
• [Publications] 江橋 節郎: "無機イオンと蛋白質と生命" 薬学雑誌. 111. 182-187 (1991)
• [Publications] 江橋 節郎: "Dimethyl sulfoxide (DMSO)による蛋白質分画法" 生化学. 371-373 (1991)
• [Publications] 八田 一郎: "モデル生体膜のトンネル顕微鏡観察" パリティ. 5. 56-57 (1991)
• [Publications] 八田 一郎: "生体膜の物性ーリン脂質膜を中心にー" 応用物理. 60. 239-246 (1991)
• [Publications] 久富 修: "PCR法の実際と視物質遺伝子クロ-ニング" 生物物理. 31. 161-163 (1991)
• [Publications] 柊 弓絃: "放射光と構造学的速度論ータバコモザイクウィルス外被タンパク質の会合過程を例として" 生化学. 63. 351-356 (1991)
• [Publications] 藤井 功: "タンパク質構造解析のモデルビルディングのためのグラフィックスプログラムの開発" 情報処理学会論文誌. 32. 599-607 (1991)
• [Publications] 江橋 節郎: "Citation Classicと私" 生物物理. 31. 48-50 (1991)
• [Publications] Ohki,K.: "Ethanol induces interdigitated gel phase(LB_1)between lamellar gel phase(Lβ')and ripple phase(Pβ')in phosphatidy lcholine membranes:a scanning density meter study." Biochem.Biophys.Acta.1028. 215-222 (1991)
• [Publications] Ono,T.: "Structural and Functional Modification of the Manganese cluster in Ca^<2+>ーDepleted SI and S_2 States:Eiectron Paramagnetic Resonance and Xーray Absorption Spectroscopy Studies" Biochemistry. 30. 6836-6841 (1991)
• [Publications] Onodera,K.: "Crystallization and preliminary Xーray studies of a Bacillus subtilis and Thermus thermophilus HB8 Chimeric 3ーisopropylmalate dehydrogenase" J.Biochem.109. 1-2 (1991)
• [Publications] Sekiya,N.: "Fourier transform infrared spectroscopic study of retinochrome and its primary photoproduct,lumiretinotinochrome" FEBS Lett.280. 107-111 (1991)
• [Publications] Shiro,Y.: "Structural and Electronic Characterization of oxygenated Hemoproteins by using XANES Spectroscopy" Biochem.Biophys.Acta.(1992)
• [Publications] Sugawar,Y.: "HumidtyーControlled Reverible Structure Transitiow of Disodium Adenosine 5'ーTriphosphate between Dihydrate and trihydrate in a single Crystal State" J.Am.chem.Soc.113. 5440-5445 (1991)
• [Publications] Takahashi,H.: "Electrostatic interaction of poly(Lーdysine)with dipalmltoyl phosphstidic acid studied Xーray diffraction" Biochem.Biophys.Acta. 1069. 229-234 (1991)
• [Publications] Takeuchi,Y.: "Refined Crystal structure of the Complex of Subtilisin BPN^+ and Streptomyces Sudtilisin Inhibitor at 1.8A" J.Mol.Biol.221. 309-325 (1991)
• [Publications] Kitagawa,Y.: "Threeーdimensional structure of Cu,ZuーSuperoxide dismutase from Spinach at 2.0 A resolution" J.Biochew.110. 472-473 (1991)
• [Publications] Konishi,M.: "Timeーresolved synchrotron Xーray diffraction studies of a single frog skeletal muscle fiber:Timeーcourses of in tensity changes of the equatorial reflections and intracellular Ca Transients" Biophys.Chem.39. 287-297 (1991)
• [Publications] Matsubara,I.: "Crossーbridge movement in fast and slow skeletal muscles of the Chick" J.Physiol.441. 113-120 (1991)
• [Publications] Mikami,T.: "Crystallization and preliminary Studies of Hydroxy lamine oxidoreductase from Nitorosomonas europaea" J.Biochem.110. 681-682 (1991)
• [Publications] Morimoto,Y.: "Effects of Amino Acid Substitwtion on ThreeーDimensional structure:An Xーray Analysis of Cytochrome C3 from Desulfovibrio vulgaris Hildenborough at 2A Resolution" J.Biochem.110. 532-540 (1991)
• [Publications] Morimoto,Y.: "Small angle Xーray scattering Study of cytochrome oxidase from Beef Heart and its complex with chtochrome c using contrast variation method" Bull.Chem.Soc.64. 1153-1159 (1991)
• [Publications] Nagamune,T.: "Crysta llization of a Photosensitive Nitrile Hydratase from Rhodococcus sp.Nー771" J.Mol.Biol.220. 221-222 (1991)
• [Publications] Nakasako,M.: "Crytallographic Choracterization of Mーintermediate Appearing in the Photocycle or Bacteriorhodopsin with Xーray Diffraction Technigue at Room Temperature" FEBS Lett.292. 73-75 (1991)
• [Publications] Iwasa,T.: "Lightーdependent proton movement and photoin termediates on bacteriorhopsin photocycle." Sci.Rep.Tohoku Univ.11. 229-237 (1991)
• [Publications] Kamiya,N.: "Heme Environmental Structure of a Novel Artificial Myoglobin With Closed Heme Pocket:SiteーSpecific chemical Modification Producing Distal NーTetrazoly lhistidine E7 by Cyanogen Bromide and Azide Iow." J.Am.Chem.Soc.113. 1826-1829 (1991)
• [Publications] Katagiri,C.: "Structure of Colorado Potato Beetle Lipophorin:Differential Scanning Calolimetric and SmallーAngle Xーray Scattering atudies" Biochemistry.30. 9675-9681 (1991)
• [Publications] Kataoka,M.: "Smallーangle Xーray Scattering Studies of Calmodwlin Mutants With Dele tions in the Linker Region of the central Helix Indicate that the Linker Region Retains a PredominantlyαーHelical conformation" Biochemistry.30. 1188-1192 (1991)
• [Publications] Kataoka,M.: "Properties of the Disorder Induced into Purple Membrane Structuve by Iodination" Sci.Rep.Tohoku Univ.11. 239-250 (1991)
• [Publications] Kataoka,M.: "Smallーangle Xーray Scattering Study of Calmodulin Bound to two Peptides Corresponding to Parts of the Calmodulinーbinding domain of the Plesma Membrane Ca^<2+>Pump" Biochemistry. 30. 6247-6251 (1991)
• [Publications] Kawaguchi,T.: "Structural Studies of a Homologous Series of AIKYI sucrose Ester Micelle by XーRay Scattering" J.Phys.Chem.95. 3837-3846 (1991)
• [Publications] Kawata,Y.: "Preliminary Xーray Crystallographic Analysis of Tryptophanase from Escherichia coli" FEBS Lett.284. 270-272 (1991)
• [Publications] 松岡 審爾: "シンクロトロン放射光による燐脂質膜の構造の研究IIー配向膜および単分子膜ー" 膜. 15. 263-268 (1990)
• [Publications] Fujii,T.: "Crystallization and preliminary Xーray studies on Sulfolobus acidocaldarius Ferredoxin" J.Biochem.110. 472-473 (1991)
• [Publications] Harada,M.: "SmallーAngle Xーray Scattering Studies of MgーAT(D)Pーinduced Hexamer Dimer Dissociation in the Reconstituted α_3β_3Complex of ATP Synthase from Thermophilic Bacterium PS3" J.Biol.Chem.266. 11455-11460 (1991)
• [Publications] Hata,T.: "The 2.0 A cdrystal structure of cyanide metmyoglobin reconstituted with 5,10,15,20ーTetrapropylhemin" Bull.Chem.Soc.64. 821-828 (1991)
• [Publications] Hiraki,K.: "Conformation Changes of Cuttlefish(Euprymna Morsei)Rhodopsin Following Photoconversion" Biochim.Biophys.Acta.1077. 355-361 (1991)
• [Publications] Hisatomi,O.: "Isolation and Characterization of lamprey rhodopsin cDNA" Biochem,.Biophys.Res.Commun.174. 1125-1132 (1991)
• [Publications] Imada,K.: "Threeーdimensional structure of a Highly Thermostable Enzyme,3ーIsopropylmalate Dehydrogenase of Thermus thermophilus at 2.2 Resolution." J.Mol,Biol.222. 725-738 (1991)
• [Publications] Inoue,H.: "structural kinetics:TemperatureーJump Synchrotron SmallーAngle Xーray Scattering study of Tobacco Mosaic Virus.Protein as an Example." Bull.Inst.Chem.Res.,Kyoto Univ.69. 92-100 (1991)
• [Publications] Takeuchi,Y.: "Molecular Recognition at the Active Site of Subtilisin BPN^+:Crystallographic Studies Using Genetically Engineered proteinaceous Inhibitor SSI(Streptomyces Subtilisin Inhibitor)" Protein Engineering. 4. 501-508 (1991)
• [Publications] Tanaka,H.: "Changes in the Xーray diffraction Pattern from rigor muscles by application of external length Changes" Adv.Biophys.27. 105-113 (1991)
• [Publications] Tokunaga,M.: "Structure and Structural Change of the Myosin Head." Adv.Biophys.27. 157-167 (1991)
• [Publications] Uruga,T.: "Effects of volatile Anesthietcs on Bacteriorhodopsin in Purple Membrane,Halobacterium Halobiumcells and Reconstitwted Vesicles" Biophys.Chem.41. 157-168 (1991)
• [Publications] Wakabayashi,k.: "Dynamic Xーray diffraction of skeletal muscle:structural change of actin filaments" Adv.Biophys.27. 3-12 (1991)
• [Publications] Yagi,N.: "Intensification of the first actin layerーline during contraction of frog skeletal muscle." Adv.Biophys.27. 35-43 (1991)
• [Publications] Yamashita,I.: "Preparing Wellーoriented Sols of Straight Bacterial Flagellar Filaments for Xーray Fiber Diffraction" J.Mol.Biol.217. 293-302 (1991)
• [Publications] Yao,H.: "Metastable ripple phase of fully hydrated dipalmitoyl Phosphatidylcholine as Studied by Small angle Xーray scattering" Biophys.J.59. 252-255 (1991)
• [Publications] Ebashi,S.: "Studies on ca^<2+> regulation of muscle contraction:Pastand Present New trends in biological chemistry" Japan Sci.Press,Tokyo/SpringerーVerlag,Berlin, 9 (1991)
• [Publications] Ebashi,S.: "Tropomyosin as the Spearhead of regulatory proteins,World Scientific" Retrospect and prospect of Protein research, 6 (1991)