1995 Fiscal Year Annual Research Report

タンパク質折り畳み中間体の構造と構造形成機構

Research Project

Project/Area Number	06304051
Research Institution	Osaka University
Principal Investigator	片岡幹雄大阪大学, 理学部, 助教授 (30150254)
Co-Investigator(Kenkyū-buntansha)	木原裕関西医科大学, 医学部, 教授 (20049076) 月向邦彦広島大学, 理学部, 教授 (10023467) 桑島邦博東京大学, 大学院・理学系研究科, 助教授 (70091444) 赤坂一之神戸大学, 理学部, 教授 (50025368) 後藤祐児大阪大学, 理学部, 助教授 (40153770)
Keywords	蛋白質折り畳み / モルテングロビュール / X線溶液散乱 / 蛋白質変性 / 高圧NMR / ストップトフローCD / シャペロニン / 遺伝子工学
Research Abstract	モルテングロビュール(MG)の構造形成要因が、疎水的相互作用による場合と、分子内SS結合による場合があることが明らかとなった。酸変性、熱変性及び変性剤変性状態はそれぞれに異なる構造状態であり、さらに酸変性状態を加熱するとまた別の構造をとることが示された。変異の効果が、変性構造にも現れることが示された。等温滴定が田熱量計によりMG形成反応が測定され、MG状態における疎水相互作用の程度が天然状態の40%程であることが示された。トリプトファン合成酵素αサブユニットのプロリン変異体の速度論的研究から、αサブユニットの構造形成過程には初期中間体とそれに引き続く後期中間状態が存在することが明らかとなった。プロリン残基は後期中間状態の安定化に寄与していることが示された。蛋白質溶液用高圧NMR装置が開発され、R Nase Aに応用された。R Nase Aは高圧下でも、加熱により二状態転移を行うことが判明した。また、変性による体積変化が負であること、定圧比熱変化が加圧により著しく減少することが見いだされた。シャペロニンの標的認識機構が、αラクトアルブミンのMGを用いて詳細に研究され、静電相互作用の重要性が明らかになった。 MG状態のグローバルな構造とその決定に寄与する因子、特に水の寄与についての理論的な研究が進められた。この理論に基づき、チトクロムc、ミオグロビン及びαラクトアルブミンのMG状態の構造モデルが作られ、X線溶液散乱パターンが計算された。実際のX線溶液散乱データとの比較から、この理論的方法が折り畳み研究に有望であることが示された。スタフィロコッカルヌクレアーゼのフラグメントの構造が分子動力学シミュレーションにより研究され、N末のβ構造は安定に保たれているが、C末のαヘリックス領域は完全に乱れていることが推定された。変性に伴う断熱圧縮率変化が詳細に測定され、熱変性においては二次構造の崩壊に伴いいったんコンパクトな構造に転移し、その後ゆらいだ構造になる可能性が示唆された。

Research Products

(33 results)

All Other

All Publications (33 results)

[Publications] Mikio Kataoka: "Structural characterization of molten globule and native states of apomyoglobin by solution X-ray scattering" Journal of Molecular Biology. 249. 215-228 (1995)
[Publications] Ichiro Nishii: "Thermodynamic stability of the molten globule states of apomyoglobin" Journal of Molecular Biology. 250. 223-228 (1995)
[Publications] Takashi Konno: "Small angle X-ray scattering analysis of cold-,heat-,and urea-denatured state in a protein,Streptomycessubtilisin inhibitor" Journal of Molecuar Biology. 251. 95-103 (1995)
[Publications] Yoshinobu Izumi: "The chain dynamics of mastoparan bound to calmodulin" Physica. B213&214. 772-774 (1995)
[Publications] Yasushi Imamoto: "Reconstitution of photoactive yellow protein from apoprotein and p-coumaric acid deribatives" FEBS Letters. 374. 157-160 (1995)
[Publications] 片岡幹雄: "タンパク質のX線溶液散乱" 生物物理. 35. 239-241 (1995)
[Publications] Daizo Hamada: "Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c" Journal of Molecular Biology. 256. 172-186 (1996)
[Publications] Mikio Kataoka: "The linker of calmodulin lacking Glu84 is elongated in solution;Although it is bent in the crystal" Proteins:Structure,Function and Genetetics. (印刷中). (1996)
[Publications] Hironari Kamikubo: "Structure of the N intermediate of bacteriorhodopsin revealed by X-ray diffraction" Proceedings of National Academy of Science,U.S.(印刷中). (1996)
[Publications] Lishi: "Conformational characterization of Dnak and its complexes with substrate protein by small-angle X-ray scatterig" Biochemistry. (印刷中). (1996)
[Publications] Kentaro Shiraki: "Trifluoroethanol-induced stabilization of the α-helical structure of β-lactoglobulin:Implication for non-hierarchical protein folding" Journal of Molecular Biology. 237. 336-348 (1995)
[Publications] Daizo Hama: "Salt-induced formation of the molten globule state of apomyoglobin studied by isothermal titration calorimetry" Thermochica Acta. 226. 385-400 (1995)
[Publications] Daizo Hamada: "High helical propensity of the peptide fragments derived from β-lactoglobulin,a predominantly β-sheet protein:Implication for non-hierarchical protein folding" Journal of Molecular Biology. 254. 737-746 (1995)
[Publications] Kouhei Tsumoto: "Role of Tyr residues in the contact region of anti-lysozyme monoclonal antibody HyHEL10 for antigen binding" Journal of Biological Chemistry. 270. 18551-18557 (1995)
[Publications] 油谷克英: "タンパク質の変性の熱力学" 生物物理. 35. 3-7 (1996)
[Publications] Kazufumi Takano: "Contribution of hydrophobic residues to the stability of human lysozyme:Calorimetric studies and X-ray structural analysis of the five Ile to Valmutants" Journal of Molecular Biology. 254. 62-76 (1996)
[Publications] Kazuhiko Yamazaki: "Folding pathway of Escherichia coli ribonuclease HI:A circular dichroism,fluorescence,and NMR study" Biochemistry. 34. 16552-16562 (1995)
[Publications] Youjiro Tamura: "Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility" Biochemistry. 34. 1878-1884 (1995)
[Publications] 月向邦彦: "密度と部分比容の測定" 蛋白質・核酸・酵素. 40. 2461-2465 (1995)
[Publications] Kunihiko Gekko: "A large compressibility change of protein induced by a single amino acid substitution" Protein Science. 5(印刷中). (1996)
[Publications] Eiji Ohmae: "Effects of point mutation at a flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on the stability and function" Journal of Biochemistry. (印刷中). (1996)
[Publications] Mamoru Sato: "The ATP-hydrolyzing excited structure of the reconstituted α3β3 complex of ATP synthase from thermophilic bacterium PS3:the structural feature revealed by time-resolved small-angle x-ray scattering with synehrotron radiation" Journal of Biochemistry. 117. 113-119 (1995)
[Publications] Yoshihiko Igarashi: "Solution X-ray scattering study on the chaperonin GroEL for Escherichia coli" Biophysical Chemistry. 53. 259-266 (1995)
[Publications] N.N.Kalnin: "Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism" Proteins:Structure,Functionh,Genetics. 23. 163-176 (1995)
[Publications] Akira Okazaki: "What is the molten globule?Reply" Nature Structure Biology. 2. 10-11 (1995)
[Publications] Hidefumi Uchiyama: "Effects of amino acid substitutions in the hydrophobic core of α-lactalbumin on the stability of the molten globule state" Protein Engineering. (印刷中). (1995)
[Publications] Kunihiro Kuwajima: "The molten globule state of α-lactalbumin" FASEB Journal. 10. 102-109 (1996)
[Publications] Kumiko Katsumata: "Effect of GroEL on the refolding kinetics of α-lactalbumin" Journal of Molecular Biology. (印刷中). (1996)
[Publications] 桑島邦博: "GroELによる標的タンパク質認識の分子機構" 蛋白質・核酸・酵素. (印刷中). (1996)
[Publications] Noriyuki Yamaotsu: "Solvation free energies of amino acids calculated by molecular dynamics/free energy perturbation method" Chemical Pharmacology Bullettin. 43. 717-721 (1995)
[Publications] Tohru Yamaguchi: "Thermodynamics of unfolding of ribonuclease A under high pressure.A study by Proton NMR" Journal of Molecular Biology. 250. 689-694 (1995)
[Publications] Atsuo Tamura: "Dynamics of the three methionyl side chains of Streptomyces subtilisininhibitor Deuterium NMR studies in solution and in solid" Protein Science. 5. 127-139 (1996)
[Publications] 曽田邦嗣: "タンパク質の構造安定性の熱力学と疎水効果" ぶんせき. 1995(8). 630-637 (1995)

1995 Fiscal Year Annual Research Report

タンパク質折り畳み中間体の構造と構造形成機構

Principal Investigator

片岡 幹雄 大阪大学, 理学部, 助教授 (30150254)

Research Products

[Publications] Mikio Kataoka: "Structural characterization of molten globule and native states of apomyoglobin by solution X-ray scattering" Journal of Molecular Biology. 249. 215-228 (1995)

[Publications] Ichiro Nishii: "Thermodynamic stability of the molten globule states of apomyoglobin" Journal of Molecular Biology. 250. 223-228 (1995)

[Publications] Takashi Konno: "Small angle X-ray scattering analysis of cold-,heat-,and urea-denatured state in a protein,Streptomycessubtilisin inhibitor" Journal of Molecuar Biology. 251. 95-103 (1995)

[Publications] Yoshinobu Izumi: "The chain dynamics of mastoparan bound to calmodulin" Physica. B213&214. 772-774 (1995)

[Publications] Yasushi Imamoto: "Reconstitution of photoactive yellow protein from apoprotein and p-coumaric acid deribatives" FEBS Letters. 374. 157-160 (1995)

[Publications] 片岡幹雄: "タンパク質のX線溶液散乱" 生物物理. 35. 239-241 (1995)

[Publications] Daizo Hamada: "Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c" Journal of Molecular Biology. 256. 172-186 (1996)

[Publications] Mikio Kataoka: "The linker of calmodulin lacking Glu84 is elongated in solution;Although it is bent in the crystal" Proteins:Structure,Function and Genetetics. (印刷中). (1996)

[Publications] Hironari Kamikubo: "Structure of the N intermediate of bacteriorhodopsin revealed by X-ray diffraction" Proceedings of National Academy of Science,U.S.(印刷中). (1996)

[Publications] Lishi: "Conformational characterization of Dnak and its complexes with substrate protein by small-angle X-ray scatterig" Biochemistry. (印刷中). (1996)

[Publications] Kentaro Shiraki: "Trifluoroethanol-induced stabilization of the α-helical structure of β-lactoglobulin:Implication for non-hierarchical protein folding" Journal of Molecular Biology. 237. 336-348 (1995)

[Publications] Daizo Hama: "Salt-induced formation of the molten globule state of apomyoglobin studied by isothermal titration calorimetry" Thermochica Acta. 226. 385-400 (1995)

[Publications] Daizo Hamada: "High helical propensity of the peptide fragments derived from β-lactoglobulin,a predominantly β-sheet protein:Implication for non-hierarchical protein folding" Journal of Molecular Biology. 254. 737-746 (1995)

[Publications] Kouhei Tsumoto: "Role of Tyr residues in the contact region of anti-lysozyme monoclonal antibody HyHEL10 for antigen binding" Journal of Biological Chemistry. 270. 18551-18557 (1995)

[Publications] 油谷克英: "タンパク質の変性の熱力学" 生物物理. 35. 3-7 (1996)

[Publications] Kazufumi Takano: "Contribution of hydrophobic residues to the stability of human lysozyme:Calorimetric studies and X-ray structural analysis of the five Ile to Valmutants" Journal of Molecular Biology. 254. 62-76 (1996)

[Publications] Kazuhiko Yamazaki: "Folding pathway of Escherichia coli ribonuclease HI:A circular dichroism,fluorescence,and NMR study" Biochemistry. 34. 16552-16562 (1995)

[Publications] Youjiro Tamura: "Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility" Biochemistry. 34. 1878-1884 (1995)

[Publications] 月向邦彦: "密度と部分比容の測定" 蛋白質・核酸・酵素. 40. 2461-2465 (1995)

[Publications] Kunihiko Gekko: "A large compressibility change of protein induced by a single amino acid substitution" Protein Science. 5(印刷中). (1996)

[Publications] Eiji Ohmae: "Effects of point mutation at a flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on the stability and function" Journal of Biochemistry. (印刷中). (1996)

[Publications] Yoshihiko Igarashi: "Solution X-ray scattering study on the chaperonin GroEL for Escherichia coli" Biophysical Chemistry. 53. 259-266 (1995)

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

[Publications] Akira Okazaki: "What is the molten globule?Reply" Nature Structure Biology. 2. 10-11 (1995)

[Publications] Hidefumi Uchiyama: "Effects of amino acid substitutions in the hydrophobic core of α-lactalbumin on the stability of the molten globule state" Protein Engineering. (印刷中). (1995)

[Publications] Kunihiro Kuwajima: "The molten globule state of α-lactalbumin" FASEB Journal. 10. 102-109 (1996)

[Publications] Kumiko Katsumata: "Effect of GroEL on the refolding kinetics of α-lactalbumin" Journal of Molecular Biology. (印刷中). (1996)

[Publications] 桑島邦博: "GroELによる標的タンパク質認識の分子機構" 蛋白質・核酸・酵素. (印刷中). (1996)

[Publications] Noriyuki Yamaotsu: "Solvation free energies of amino acids calculated by molecular dynamics/free energy perturbation method" Chemical Pharmacology Bullettin. 43. 717-721 (1995)

[Publications] Tohru Yamaguchi: "Thermodynamics of unfolding of ribonuclease A under high pressure.A study by Proton NMR" Journal of Molecular Biology. 250. 689-694 (1995)

[Publications] Atsuo Tamura: "Dynamics of the three methionyl side chains of Streptomyces subtilisininhibitor Deuterium NMR studies in solution and in solid" Protein Science. 5. 127-139 (1996)

[Publications] 曽田邦嗣: "タンパク質の構造安定性の熱力学と疎水効果" ぶんせき. 1995(8). 630-637 (1995)

片岡幹雄大阪大学, 理学部, 助教授 (30150254)