1995 Fiscal Year Annual Research Report

モジュールの組み合わせによるタンパク質設計法の開発

Research Project

Project/Area Number	06558100
Research Institution	Nagoya University
Principal Investigator	郷通子名古屋大学, 理学部, 教授 (70037290)
Co-Investigator(Kenkyū-buntansha)	桑島邦博東京大学, 大学院・理学系研究科, 助教授 (70091444) 北絛裕信大阪市立大学, 工学部, 講師 (00209214) 野口俊之名古屋大学, 理学部, 助教授 (90172775)
Keywords	ミニバルナーゼ / モジュール / RNA分解活性 / モルテングロビュール / タンパク質デザイン / 固相法 / セグメント縮合 / チオエステル
Research Abstract	モジュールを単位とした新しいタンパク質の設計法を実用化するための技術を確立する事をめざした。目標は(1)天然のタンパク質からモジュール単位の削除を行い、安定な立体構造をもつミニタンパク質を設計、それを作成して構造解析を行うこと、(2)親タンパク質の機能を保持したミニタンパク質の設計、作成、構造と機能の測定である。2年間で以下の実績が得られた。 1)基本構造の設計と安定性の評価:モジュールを削除したM1欠失バルナーゼとM2欠失バルナーゼが目的に合うことを見出した。ミニ版安定化の実現に必要なアミノ酸置換の位置と置換後のアミノ酸種類を選定した。分子動力学計算と自由エネルギー変化の計算により、ミニバルナーゼの構造安定性を評価した。 2)ミニタンパク質の化学合成:北絛らによって独自に開発されたタンパク質合成法を用い、アミノ酸配列をセグメントに分割し、固相法により合成した。この際、末端カルボキシル基をチオエステル基に置き換えた。ラベルしたアミノ酸は手動で、それ以外のアミノ酸は自動合成機にて縮合した。 3)ミニバルナーゼの構造と活性の測定:ラベルしたアミノ酸のケミカルシフトからM2欠失バルナーゼが安定な3次構造を形成している事が判明した。また、M1欠失バルナーゼはモジュールM1と複合体を形成し、RNA分解活性をもつことが分かった。以上、ミニタンパク質デザインのための構造安定化技術と合成法を確立することができた。今後、ミニバルナーゼの3次構造をNMR法により決定し、バルナーゼの立体構造との違いを明白にする。構造安定化に加えて活性をもつミニバルナーゼをデザインする技術を開発する事が今後の課題である。

Research Products
(15 results)

All Other

All Publications (15 results)

[Publications] Nakashima,T.: "Structure of the human CCG1 gene;relationship between the exons/introns and functional domain/modules of the protein" Gene. 141. 193-200 (1994)
[Publications] Hojo,H.: "Preparation of S-protected cysteine-containing peptide thioester and its use for the synthesis of the barnase-like domain in DNA-directed RNA polymerase II of Saccharomvces cerevisiae" Bull.Chem.Soc.Japan. 68. 330-336 (1995)
[Publications] Noguti,T.: "Insect prothoracicotropic hormone:A new member of the vertebrate growth factor superfamily" FEBS Lett.376. 251-256 (1995)
[Publications] Tateno,M.: "Module Structure and Function of Glutamyl-tRNA Synthetase" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 53-63 (1995)
[Publications] Noguti,T.: "Modules of barnase:the physicochemical basis for their structures" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 161-174 (1995)
[Publications] Takahashi,K.: "Mechanical stability of protein modules determined by molecular dynamics simulations" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 175-185 (1995)
[Publications] Yura,K.: "Helix-turn-helix module distribution and module shuffling" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 187-195 (1995)
[Publications] Oshima,T.: "Module replacement converted coenzyme specificity of isocitrate dehydrogenase" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 197-203 (1995)
[Publications] Go,M.: "Putative origin of introns deduced from protein anatomy" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 229-235 (1995)
[Publications] Fukami-Kobayashi,K.: "Correlation between module boundaries and intron positions in hemoglobins from various taxa" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 271-282 (1995)
[Publications] Ogata,K.: "Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb" Nature Structural Biology. 2. 309-320 (1995)
[Publications] Kalnin,N.: "Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism" Proteins:Structure,Function,and Genetics. 23. 163-176 (1995)
[Publications] Uchiyama,H.: "Effects of amino acid substitutions in the hydrophobic core of alpha-lactoalbumin on the stability of the molten globule state" Protein Engineering. (in press). (1995)
[Publications] Kuwajima,K.: "The molten globule state of alpha-lactoalbumin" The FASEB journal. 10. 102-109 (1996)
[Publications] Song,S.: "Cloning and characterzation of the gene encoding Halobacterium halobium adenylate kinase(in press)" Gene.

1995 Fiscal Year Annual Research Report

モジュールの組み合わせによるタンパク質設計法の開発

Principal Investigator

郷 通子 名古屋大学, 理学部, 教授 (70037290)

Research Products

[Publications] Nakashima,T.: "Structure of the human CCG1 gene;relationship between the exons/introns and functional domain/modules of the protein" Gene. 141. 193-200 (1994)

[Publications] Hojo,H.: "Preparation of S-protected cysteine-containing peptide thioester and its use for the synthesis of the barnase-like domain in DNA-directed RNA polymerase II of Saccharomvces cerevisiae" Bull.Chem.Soc.Japan. 68. 330-336 (1995)

[Publications] Noguti,T.: "Insect prothoracicotropic hormone:A new member of the vertebrate growth factor superfamily" FEBS Lett.376. 251-256 (1995)

[Publications] Tateno,M.: "Module Structure and Function of Glutamyl-tRNA Synthetase" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 53-63 (1995)

[Publications] Noguti,T.: "Modules of barnase:the physicochemical basis for their structures" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 161-174 (1995)

[Publications] Takahashi,K.: "Mechanical stability of protein modules determined by molecular dynamics simulations" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 175-185 (1995)

[Publications] Yura,K.: "Helix-turn-helix module distribution and module shuffling" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 187-195 (1995)

[Publications] Oshima,T.: "Module replacement converted coenzyme specificity of isocitrate dehydrogenase" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 197-203 (1995)

[Publications] Go,M.: "Putative origin of introns deduced from protein anatomy" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 229-235 (1995)

[Publications] Fukami-Kobayashi,K.: "Correlation between module boundaries and intron positions in hemoglobins from various taxa" In Tracing Biological Evolution in Protein and Gene Structures(Eds.M.Go & P.Schimmel)Elsevier Science. 271-282 (1995)

[Publications] Ogata,K.: "Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb" Nature Structural Biology. 2. 309-320 (1995)

[Publications] Kalnin,N.: "Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism" Proteins:Structure,Function,and Genetics. 23. 163-176 (1995)

[Publications] Uchiyama,H.: "Effects of amino acid substitutions in the hydrophobic core of alpha-lactoalbumin on the stability of the molten globule state" Protein Engineering. (in press). (1995)

[Publications] Kuwajima,K.: "The molten globule state of alpha-lactoalbumin" The FASEB journal. 10. 102-109 (1996)

[Publications] Song,S.: "Cloning and characterzation of the gene encoding Halobacterium halobium adenylate kinase(in press)" Gene.

郷通子名古屋大学, 理学部, 教授 (70037290)