時間分割蛋白質結晶構造解析法の研究

1996 Fiscal Year Annual Research Report

• Project/Area Number: 05244102
• Research Institution: Nagoya University
• Principal Investigator: 山根 隆 名古屋大学, 工学部, 教授 (80030055)
• Co-Investigator(Kenkyū-buntansha): 三木 邦夫 京都大学, 大学院・理学研究科, 教授 (10116105) ; 月原 富武 大阪大学, 蛋白質研究所, 教授 (00032277) ; 田中 信夫 東京工業大学, 生命理工学部, 教授 (50032024) ; 佐藤 能雅 東京大学, 薬学部, 教授 (30150014) ; 安岡 則武 姫路工業大学, 理学部, 教授 (40029054)
• Keywords: 蛋白質結晶 / 動的解析 / ラウエ法 / 放射光 / 酵素反応トレース / 良質結晶作成法 / 温度ジャンプ / pHジャンプ

Research Abstract

本重点領域研究では、蛋白質結晶学における通常の静的な構造解析を越える動的な解析を目指して、研究分担者が、以下の役割を担当した。得られた研究成果は、得られた研究成果は、当重点領域研究のNews Letterや公開シンポジウムで発表された。主要な研究実績は以下の通りである。山根:「酵素反応のトレース」で、「フローセルを用いたラウエ法の加水分解酵素への適用」で、M-プロテアーゼのアルカリ安定化機構を解明した。「モジュール置換キメラへモグロビンの結晶構造解析」で大希望に改変した蛋白質の構造と機能を解明した。安岡:「電子伝達反応の研究」でチトクロムc'のヘムに配位子を結合させ、ヘム周辺のアミノ酸側鎖の移動を観察し、機能の解明を進めた。佐藤:「酵素反応トレース」で「糖蛋白質ヒト腎臓ジペプチダーゼの低温結晶構造解析」を行い、活性発現機構を検討した。田中:赤外線ランプおよびレーザー用いる2種類の温度ジャンプ法の開発に成功した。「酵素耐熱化機構の研究」でレーザー照射前後でのイソプロピルリンゴ酸脱水素酵素の構造変化を観測し、耐熱性の検討を行った。月原:「良質結晶作成法の開発、分子置換法解析法の開発」で超分子複合体であるチトクロム酸化酵素の良質の結晶を調整し、2.8A分解能の結晶構造を世界で初めて解明した。三木:「良質結晶作成法の開発、結晶評価法の開発」で、結晶化が極めて困難な膜蛋白質である光合成反応中心複合体の結晶化など多数の蛋白質の結晶化に成功した。松浦:結晶化に用いる沈殿剤の役割と結晶成長との関係を検討した。箱島「実験室系でのX線強度測定系の構築」を行い、性能を評価した。相原:微小重力場では結晶成長過程に違いのあることを示した。井上:「電子伝達酵素反応のトレース」でプラストシアニンの動的解析のためにラウエ法データで構造解析を行い、単色光データと同程度の精密な分子の立体構造を決定した。

Research Products

• [Publications] A.Suzuki,T.Yamane,T.Ashida: "Crystallographic refinement of Bowman-Birk type protease inhibitor A-II from Peanut (Arachis hypogaea) at 2.3A resolution." J.MOl.Biol.234. 722-734 (1993)
• [Publications] A.Suzuki,T.Yamane,T.Ashida: "Crystallization and preliminary X-ray studies on the trypsin inhibitor I-2 from wheat germ and its complex with trypsin." Acta Crystallogr.D49. 594-596 (1993)
• [Publications] T.Yamane,A.Suzuki,T.Ashida: "Crystal structure of a new aikaline serine protease (M-protease) from Bacillus sp.KSM-K16" Acta Crystallogr.D51. 199-206 (1995)
• [Publications] A.Suzuki,T.Yamane,T.Ashida: "Crystal structure analysis of phospholipase A2 from Trimeresurus flavoviridis (Habu snake) venom at 1.5A resolution." J.Biochem.117. 730-740 (1995)
• [Publications] T.Yamane,A.Suzuki,A.Ashida: "Crystal structure of Streptomyces erythraeus trypsin at 1.9A resolution." J.Biochem.118. 882-894 (1995)
• [Publications] T.Shirai,A.Suzuki,T.Yamane,T.Ashida: "High-resolution crystal structure of M-protease : phylogeny aided analysis of the high-alkaline adaptation mechanism." Protein Engineering. (in press). (1997)
• [Publications] Y.Higuchi,N.Yasuoka,T.E.Meyer,M.A.Cusanovich: "Crystallization and preliminary X-ray study of a new crystal form of cytochrome c' from Rhodobacter capsulatus" Acta Cryst.D52. 199-200 (1996)
• [Publications] T.H.Tahirov,S.Misakai,Y.Higuchi,N.Yasuoka: "High-resolution crystal structures of two polymorphs of cytochrome c' from purple phototrophic bacterium Rhodobacter capsulatus" J.Mol.Biol.259. 467-479 (1996)
• [Publications] T.H.Tahirov,S.Misakai,T.E.Meyer,N.Yasuoka: "Concerned movement of side chains in the haem vicinity observed on ligand binding in cytochrome c' from Rhodobacter capsulatus" Nature Structural Biology. 3. 459-464 (1996)
• [Publications] S.Noguchi,Y.Satow,C.Sasaki,T.Matsuzaki: "Crystal Stracture of Ustilago shpaerogena Ribonuclease U2 at 1.8Å Resulution" Biochemistry. 34. 15583-15591 (1995)
• [Publications] S.Noguchi,Y.Satow,C.Sasaki,T.Matsuzaki: "Crystallization and preliminary X-ray investigatio of six new crystal forms of ribonuclease U2 from Ustilago sphaerogena" Journal of Crystal Growth. 168. 270-274 (1996)
• [Publications] Y.Nitanai,Y.Satow,H.Adachi,M.Tsujimoto: "Cyrstallization and preliminary X-ray investigation of a glycoprotein,human renal dipeptidase" Journal of Crystal Growth. 168. 280-283 (1996)
• [Publications] M.Nishida,S.Harada,Y.Satow,K.Takahashi: "Crystallization and X-ray diffraction studies of glutathione S-transferase from Escherichia coli" Journal of Crystal Growth. 168. 284-287 (1996)
• [Publications] K.Miyawaki,S.Noguchi,S.Harada,Y.Satow: "Crystallography of succinimide hen egg-white lysozyme at low temperatures" Journal of Crystal Growth. 168. 292-296 (1996)
• [Publications] M.Sakurai,M.Ohzeki,N.Tanaka,T.Oshima: "Structure of a Loop-Deleted Variant of 3-Isopropylmalate Dehydrogenase from Thermus thermophilus : an Internal Reprieve Tolerance Mechanism" Acta Cryst..D52. 124-128 (1996)
• [Publications] C.Nagata,H.Moriyama,N.Tanaka,T.Oshima: "Crystallography of 3-Isopropylmalate Dehydrogenase from Thermus the rmophillus and its Chimeric Enzyme" Acta Cryst..D52. 623-630 (1996)
• [Publications] N.Igarashi,H.Moriyama,T.Mikami and N.Tanaka: "Detwinning of hemihedrally twinned crystals by the least squares method and its application to the crystal of hydroxylamine oxidoreductase from Nitrosomonas europaea" To be pubished.
• [Publications] T.Tsukihara,H.Aoyama,R.Yaono,S.Yoshikawa: "The Whole Structure of the 13-Subunit Oxidized Cytochrome c Oxidase at 2.8Å" Science. 272. 1136-1144 (1996)
• [Publications] K.Miki,S.Sogabe,A.Uno,T.Ezoe,N.Kasai: "Application of automatic molecular replacement procedure to crystal structure analysis of cytochrome c2 from R.viridis" Acta Crystallogr.D50. 271-275 (1994)
• [Publications] S.Sogabe,T.Ezoe,N.Kasai,M.Saeda & K.Miki: "Structural similarity of cytochrome c2 from R.viridis to mitochondrial cytochrome c reversed by its crystal structure" FEBS Lett.345. 5-8 (1994)
• [Publications] N.Katayama,M.Kobayasi F.Motojima,K.Miki: "Preliminary X-ray crystallographic studies of photosynthetic reaction center from a thermophilic sulfur bacterium,C.tepidum" FEBS Lett.348. 158-160 (1994)
• [Publications] K.Saika,K.Inaka,T.Matsui,K.Miki: "Crystallization of mutant β subunit of F1-ATPase from thermophilic Bacillus PS3" J.Mol.Biol.242. 709-711 (1994)
• [Publications] A.Kita,S.Kasai,K.Miki: "Crystal structure determination of a flavoprotein FP390 from a luminescent bacterium,P.phosphoreum" J.Biochem.117. 575-578 (1995)
• [Publications] H.Nishida,K.Inaka,K.Miki: "Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4A resolution" Biochemistry. 34. 2763-2767 (1995)
• [Publications] H.Nishida,K.Inaka & K.Miki: "Specific arragement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase" FEBS Lett.361. 97-100 (1995)
• [Publications] T.Tamada,H.Nishida,K.Miki: "A new crystal form of photolyase (photoreactivating enzyme) from the cyanobacterium A.nidulans" J.Struct.Biol.115. 37-40 (1995)
• [Publications] K.Inaka,M.Kikuchi,K.Miki: "Crystal structure of a gultathionylated mutant human lysozyme : A folding intermediate mimic in the formation of a disulfide bond" Acta Crystallogr.D51. 619-625 (1995)
• [Publications] J.Saito,Y.Nagata,K.Miki: "Crystallization and preliminary X-ray crystallographic analysis of chitosanase from B.circulans MH-K1" Acta Crystallogr.D51. 856-857 (1995)
• [Publications] S.Sogabe & K.Miki: "Refined crystal structure of ferrocytochrome c2 from R.viridis at 1.6A resolution" J.Mol.Biol.252. 235-247 (1995)
• [Publications] F.Motojima,K.Inaka,K.Miki: "Preliminary X-ray diffraction studies of a new crystal form of C-1027-AG,the apoprotein of the macromolecular antitumor antibio" Acta Crystallor.D51. 1084-1085 (1995)
• [Publications] A.Kita,S.Kasai,M.Miyata & K.Miki: "The structure of flavoprotein FP390 from luminescent bacterium,P.phosphoreum at 2.7A resolution" Acta Crystallogr.D52. 77-86 (1996)
• [Publications] M.Kataoka,K.Yamamoto K.Miki: "Crystallization and preliminary X-ray diffraction study of aldehyde reductase from a red yeast,S.salmonicolor" Acta Crystallogr.D52. 405-406 (1996)
• [Publications] H.Nishida & K.Miki: "Electrostatic properties deduced from refined structures of NADH-cytochrome b5 reductase and the other flavin-dependent reductases" Protein : Structure,Function and Genetics. 26. 32-41 (1996)
• [Publications] M.Sugahara,N.Sekine-Suzuki,K.Miki: "Crystallization and preliminary X-ray diffraction studies of toxin (perfringolysin O),a pore-forming cytolysin of C.perfringens" J.Crystal Growth. 168. 288-291 (1996)
• [Publications] T.A.Funaki,Y.Takasuga,K.Miki: "Crystallization and preliminary X-ray characterization of Chaperonin 60 from P.denitrificans" J.Crystal Growth. 168. 297-300 (1996)
• [Publications] M.Tsunemi: "Crystal Structure of an Elastase-Specific Inhibitor Elafin Complexed with Porcine Pancreatic Elastase Determined at 1.9A resolution." Biochemistry. 35. 11570-11576 (1996)
• [Publications] Y.Morishita: "Crystal Structure of a Maltotetraose-forming Exo-amylase from Pseudomonas stutzeri." J.Mol.Biol.267. 1-12 (1997)
• [Publications] T.Inoue: "Crystallization and Preliminary X-Ray Studies on Pseudoazurin from Achromobacter cycloclates IAM1013" J.Biochem. 114. 761-762 (1993)
• [Publications] T.Inoue: "Refined Crystal Structure of Pseudoazurin from Methylobacterium extorquens AM1 at 1.5A Resolution." Acta Cryst.D50(3). 317-328 (1994)
• [Publications] T.Inoue: "Three-Dimensional Structure of Azurin from Achromobacter xylosoxidas NCIB11015 at 2.5A Resolution." J.Biochem.116. 1193-1197 (1994)
• [Publications] T.Kohzuma: "Spectrosoopic and Electrochemical Studies on Active-site Transitions of the Type 1 Copper Protein Pseudoazurin from Achromobacter cycloclasres." J.Biol.Chem.270. 25733-25738 (1995)
• [Publications] N.Shibata: "Orderly disposition of heterogeneous small subunits in D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach." J.Biol.Chem.271. 26449-26452 (1996)
• [Publications] N.Shibata: "Cristallization and preliminary crystalliographic studies of ribulose 1,5-bisphosphat carboxylase/oxygenase from red alga Galdieria partita with high specificity factor." J.Biochem.120. 1064-1066 (1996)
• [Publications] C.Li: "Crystallization and Preliminary X-ray Studies of Plastocyanin from Silene Expressed in E.coli 1." Acta Cryst.D53. 129-130 (1997)
• [Publications] A.Toumoto: "Preliminary X-ray studies on a new crystal form of PHO4-DNA complex." Acta Cryst.D53. 103-104 (1997)