1990 年度実績報告書

蛋白質工学の手法により生産された蛋白質の構造に関する研究

研究課題

研究課題/領域番号	63890008
研究機関	東京大学
研究代表者	太田隆久東京大学, 農学部, 教授 (30011844)
研究分担者	京極好正大阪大学, 蛋白質研究所, 教授 (90012632) 郷信広京都大学, 理学部, 教授 (50011549) 大井龍夫京都女子大学, 家政学部, 教授 (00027012) 三浦護一郎東京大学, 工学部, 教授 (30000227) 別府輝彦東京大学, 農学部, 教授 (80011873)
キーワード	蛋白質工学 / 蛋白質の構造形成 / フォルディング / リフォルディング / NMR / 蛋白質構造のエネルギ-
研究概要	蛋白質の構造解析についてはリゾチ-ムについて特に非線形的動きを基準振動変数の動きとして記述する方法を開発し、メリチンを用いた解析により蛋白質中の低振動の協奏的な運動に及ぼす溶媒の効果を定量的に議論する道を開き、また、基準振動変数をパラメ-タ-とする蛋白質X線結晶解析の新しいリファインメントの方法を開発した(郷)。蛋白質の立体構造の熱安定性解析のため、蛋白質の構造の水和からの寄与と分子を構成する原子間の相互作用からの寄与を原子の接触表面積の変化に比例するものとして計算する方法を確立し蛋白質の構造安定性の概略を予測することに成功した(大井)。蛋白質の溶液内での構造決定と検定については ^<13>Cー ^<15>N二重標識と全 ^<15>N標識と2次元HMQCーNOESY、3次元NOESYーHMQCによるDNA結合蛋白質CROのプロトンの帰属とNOEによる2次、3次構造情報からの構造決定に成功し(京極)、また蛋白性プロテア-ゼインヒビタ-SSIについて ^<13>Cーラベル法を用い、位置特異的アミノ酸変異体を利用することにより全カルボニル炭素の帰属を決定する方法を確立した(甲斐荘)。蛋白質工学により生産された蛋白質の構造についてはプロキモシンCR601変異体のN末端の2番目のアミノ酸のリフォ-ルディングにおける寄与をこの残基を置換することで解析し、構造の再生過程の効率にはリジン残基が深く関与していることを明かにし(別府)、ビフィズス菌のLー乳酸脱水素酵素をポリエチレングリコ-ルを用いて結晶化させ、X線結晶解を行い、アロステリック解果がサブユニット相互間の接触の変化を通じて生じ、変異酵素においてはその効果が高活性型あるいは低活性型のいずれかに固定されることで生じることを確かめた(太田)。SSIの各種変異体を用いて機能に対する構造の寄与を測定した(三浦)。

研究成果
(18件)

すべてその他

すべて文献書誌 (18件)

[文献書誌] Terada,I.: "Unique precursor structure of an extracellular protease,Aqualysin I,with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>___ー <coli>___ー." J.Biol.Chem. 265. 6576-6581 (1990)
[文献書誌] Ono,M.: "Nuculeotide sequence and characteristics of gene for Lーlactate dehydrogenase of <Thermus>___ー <aquaticus>___ー YTー1 and the deduced amino acid sequence of the enzyme." J.Biochem.107. 21-26 (1990)
[文献書誌] Koide,S.: "Crystallization of allosteric Lーlactate dehydrogenase from <Thermus>___ー <caldophilus>___ー and preliminary crystallographic data." J.Biochem.109. 6-7 (1991)
[文献書誌] 伊倉禎吉: "蛋白質の溶液構造ーNMRと距離幾何法とによるアプロ-チ" 高分子. 39. 210-203 (1990)
[文献書誌] Go,N.: "A Theorem on amplitudes of thermal atomic flucturations calculated by normal mode analysis." Biopysical Chemistry. 35. 105-112 (1990)
[文献書誌] Kidera,A.: "Refinement of Protein Dynamic Structure" Natl.Acal.Sci.USA.87. 3178-3722 (1990)
[文献書誌] Koyama,S.: "The Difference in Conformation between αーhANP,and its derivative,Met(O)ーαーhANP,in solution." Protein Engineering. 3. 393-402 (1990)
[文献書誌] Gibrat,J.F.: "Normal Mode Analysis of Human Lysozyme:Study of the Relative Motion of the Two Domains and Characterization of the Harmonic Motion." Proteins. 8. 258-279 (1990)
[文献書誌] Serikawa,K.: "Efficient Expression and Zn(II)ーDependent stucture of the DNA Binding Domain of the Yeast GAL4 Protein." Protein Engineering. 3. 267-272 (1990)
[文献書誌] Sirakawa,M.: "Interaction of λーCro Repressor protein with Operator DNA Fragments Monitored as to Amide Proton Magnetic Resonances." J.Mol.Str.242. 355-366 (1991)
[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)
[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of the streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)
[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)
[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Altereation of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)
[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis I.Dynamics of Globin and the Heme Globin Interaction" J.Mol.Biol.216. 95-109 (1990)
[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis II.The conformational Change upon Oxygenation" J.Mol.Biol.216. 111-126 (1990)
[文献書誌] Nakashima,H.: "Distinct Character in Hydrophobicity of Amino Compositions of Mitochondrial Proteins." Proteins:Structure,Function,and Genentics. 8. 173-178 (1990)
[文献書誌] Ikura,T.: "Refined Structure of Melittin Bound to Perdeuterated Dodecylphosphoーcholine Micelles as Studied by 2DーNMR and Distance Geometry Galculation." Proteins. 9. 81-89 (1991)

1990 年度実績報告書

蛋白質工学の手法により生産された蛋白質の構造に関する研究

研究代表者

太田隆久東京大学, 農学部, 教授 (30011844)

研究成果

[文献書誌] Terada,I.: "Unique precursor structure of an extracellular protease,Aqualysin I,with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>_ー <coli>_ー." J.Biol.Chem. 265. 6576-6581 (1990)

[文献書誌] Ono,M.: "Nuculeotide sequence and characteristics of gene for Lーlactate dehydrogenase of <Thermus>_ー <aquaticus>_ー YTー1 and the deduced amino acid sequence of the enzyme." J.Biochem.107. 21-26 (1990)

[文献書誌] Koide,S.: "Crystallization of allosteric Lーlactate dehydrogenase from <Thermus>_ー <caldophilus>_ー and preliminary crystallographic data." J.Biochem.109. 6-7 (1991)

[文献書誌] 伊倉禎吉: "蛋白質の溶液構造ーNMRと距離幾何法とによるアプロ-チ" 高分子. 39. 210-203 (1990)

[文献書誌] Go,N.: "A Theorem on amplitudes of thermal atomic flucturations calculated by normal mode analysis." Biopysical Chemistry. 35. 105-112 (1990)

[文献書誌] Kidera,A.: "Refinement of Protein Dynamic Structure" Natl.Acal.Sci.USA.87. 3178-3722 (1990)

[文献書誌] Koyama,S.: "The Difference in Conformation between αーhANP,and its derivative,Met(O)ーαーhANP,in solution." Protein Engineering. 3. 393-402 (1990)

[文献書誌] Gibrat,J.F.: "Normal Mode Analysis of Human Lysozyme:Study of the Relative Motion of the Two Domains and Characterization of the Harmonic Motion." Proteins. 8. 258-279 (1990)

[文献書誌] Serikawa,K.: "Efficient Expression and Zn(II)ーDependent stucture of the DNA Binding Domain of the Yeast GAL4 Protein." Protein Engineering. 3. 267-272 (1990)

[文献書誌] Sirakawa,M.: "Interaction of λーCro Repressor protein with Operator DNA Fragments Monitored as to Amide Proton Magnetic Resonances." J.Mol.Str.242. 355-366 (1991)

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of the streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)

[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)

[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Altereation of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)

[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis I.Dynamics of Globin and the Heme Globin Interaction" J.Mol.Biol.216. 95-109 (1990)

[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis II.The conformational Change upon Oxygenation" J.Mol.Biol.216. 111-126 (1990)

[文献書誌] Nakashima,H.: "Distinct Character in Hydrophobicity of Amino Compositions of Mitochondrial Proteins." Proteins:Structure,Function,and Genentics. 8. 173-178 (1990)

[文献書誌] Ikura,T.: "Refined Structure of Melittin Bound to Perdeuterated Dodecylphosphoーcholine Micelles as Studied by 2DーNMR and Distance Geometry Galculation." Proteins. 9. 81-89 (1991)

1990 年度 実績報告書

蛋白質工学の手法により生産された蛋白質の構造に関する研究

研究代表者

太田 隆久 東京大学, 農学部, 教授 (30011844)

研究成果

[文献書誌] Terada,I.: "Unique precursor structure of an extracellular protease,Aqualysin I,with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>___ー <coli>___ー." J.Biol.Chem. 265. 6576-6581 (1990)

[文献書誌] Ono,M.: "Nuculeotide sequence and characteristics of gene for Lーlactate dehydrogenase of <Thermus>___ー <aquaticus>___ー YTー1 and the deduced amino acid sequence of the enzyme." J.Biochem.107. 21-26 (1990)

[文献書誌] Koide,S.: "Crystallization of allosteric Lーlactate dehydrogenase from <Thermus>___ー <caldophilus>___ー and preliminary crystallographic data." J.Biochem.109. 6-7 (1991)

[文献書誌] 伊倉 禎吉: "蛋白質の溶液構造ーNMRと距離幾何法とによるアプロ-チ" 高分子. 39. 210-203 (1990)

[文献書誌] Go,N.: "A Theorem on amplitudes of thermal atomic flucturations calculated by normal mode analysis." Biopysical Chemistry. 35. 105-112 (1990)

[文献書誌] Kidera,A.: "Refinement of Protein Dynamic Structure" Natl.Acal.Sci.USA.87. 3178-3722 (1990)

[文献書誌] Koyama,S.: "The Difference in Conformation between αーhANP,and its derivative,Met(O)ーαーhANP,in solution." Protein Engineering. 3. 393-402 (1990)

[文献書誌] Gibrat,J.F.: "Normal Mode Analysis of Human Lysozyme:Study of the Relative Motion of the Two Domains and Characterization of the Harmonic Motion." Proteins. 8. 258-279 (1990)

[文献書誌] Serikawa,K.: "Efficient Expression and Zn(II)ーDependent stucture of the DNA Binding Domain of the Yeast GAL4 Protein." Protein Engineering. 3. 267-272 (1990)

[文献書誌] Sirakawa,M.: "Interaction of λーCro Repressor protein with Operator DNA Fragments Monitored as to Amide Proton Magnetic Resonances." J.Mol.Str.242. 355-366 (1991)

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of the streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)

[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)

[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Altereation of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)

[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis I.Dynamics of Globin and the Heme Globin Interaction" J.Mol.Biol.216. 95-109 (1990)

[文献書誌] Seno,Y.: "Deoxymyoglobin Studied by the Conformational Normal Mode Analysis II.The conformational Change upon Oxygenation" J.Mol.Biol.216. 111-126 (1990)

[文献書誌] Nakashima,H.: "Distinct Character in Hydrophobicity of Amino Compositions of Mitochondrial Proteins." Proteins:Structure,Function,and Genentics. 8. 173-178 (1990)

[文献書誌] Ikura,T.: "Refined Structure of Melittin Bound to Perdeuterated Dodecylphosphoーcholine Micelles as Studied by 2DーNMR and Distance Geometry Galculation." Proteins. 9. 81-89 (1991)

1990 年度実績報告書

太田隆久東京大学, 農学部, 教授 (30011844)

[文献書誌] Terada,I.: "Unique precursor structure of an extracellular protease,Aqualysin I,with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>_ー <coli>_ー." J.Biol.Chem. 265. 6576-6581 (1990)

[文献書誌] Ono,M.: "Nuculeotide sequence and characteristics of gene for Lーlactate dehydrogenase of <Thermus>_ー <aquaticus>_ー YTー1 and the deduced amino acid sequence of the enzyme." J.Biochem.107. 21-26 (1990)

[文献書誌] Koide,S.: "Crystallization of allosteric Lーlactate dehydrogenase from <Thermus>_ー <caldophilus>_ー and preliminary crystallographic data." J.Biochem.109. 6-7 (1991)

[文献書誌] 伊倉禎吉: "蛋白質の溶液構造ーNMRと距離幾何法とによるアプロ-チ" 高分子. 39. 210-203 (1990)