1989 Fiscal Year Annual Research Report

膜蛋白質の構造決定因子の解明と結晶化技術の開発

Research Project

Project/Area Number	01304061
Research Institution	The Institute of Physical and Chemical Research
Principal Investigator	神山勉理化学研究所, 生物物理研究室, 研究員 (30170210)
Co-Investigator(Kenkyū-buntansha)	三木邦夫大阪大学, 工学部, 助手 (10116105) 白木原康雄東京大学, 理学部, 助手 (20150287) 岩佐達郎東北大学, 理学部, 助手 (00133926) 富岡明宏東京大学, 工学部, 助手 (10211400) 楠見明弘東京大学, 教養部, 助教授 (50169992)
Keywords	膜蛋白質 / 蛋白質の結晶化 / ロドプシン・ファミリ- / チトクロ-ム酸化酵素 / H^+-ATPase / Ca^<++>結合膜蛋白質 / べん毛モ-タ- / 遺伝子組換え蛋白質
Research Abstract	膜蛋白質など疎水性蛋白質の結晶化法の確立を目指して、1.蛋白質の構造安定性に及ぼす諸要因の把握、2.結晶化条件の精密化、を柱に研究を行なった。研究対象としては、光合成関連蛋白質、チトクロ-ム酸化酵素、H^+-ATPase、Ca^<++>結合膜蛋白質、バクテリオロドプシンならびにロドプシン・ファミリ-の諸膜蛋白質、べん毛モ-タ-遺伝子組換え蛋白質RecA、それにアクチンを取り上げた。本研究課題の性格からいって地道な研究が主であり、顕著な成果が即座にでてきるわけではないが、研究の進歩状況は全般的にいって順調である。特に、以前から膜蛋白質の結晶化に取り組んできた班員を中心にX線解析像の記録ができるような3次元結晶が得られている。たとえばチトクロ-ム酸化酵素、H^+-ATPase、F_1およびそのサブユニットについて良質な3次元結晶が得られている。一方、個々の蛋白質の結晶化において遭遇するであろう困難を議論し、互いに啓蒙し、あるいは励まし合うという本研究課題の最大の目的に関しては、公開のシンポジウムを開催し、170名近くの聴衆とともに活発な議論を行なうことができた。特に、膜蛋白質の結晶化の一般的方法論に関する議論では、膜蛋白質の可溶化に用いる界面活性剤の種類について、あるいは脂質の除去の影響についてなど、種々の意見の交換が行われた。

Research Products
(28 results)

All Other

All Publications (28 results)

[Publications] Kouyama,T.,Kinosita,K.,Jr.,Ikegami,A.: "Correlation between internal motion and emission kinetics of tryptophan residues in proteins." Eur.J.Biochem.182. 517-521 (1989)
[Publications] Kouyama,T.,Nasuda-kouyama,A.: "Turnover rate of the proton pumping cycle of bacterorhodopsin:pH and light-intensity dependences." Biochemistry.28. 5963-5970 (1989)
[Publications] Suzuki,S.,Kawato,S.,Kouyama,T.,Kinoshita,K.,Ikegami,A.,Kawakita,M.: "Independent flexible motion of submolecular domains of the Ca^<2+>,Mg^<2+>-ATPase of sarcoplasmic reticulum measured by timeresolved fluorescence depolarization of site-specificially attched probes." Biochemistry.28. 7734-7740 (1989)
[Publications] Fukuda,K.,Ikegami,A.,Nasuda-Kouyama,A.,Kouyama,T.: "Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin." Biochemistry.
[Publications] Nasuda-Kouyama,A.,Fukuda,K.,Iio,T.,Kouyama,T.: "Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin." Biochemistry.
[Publications] Akihiro Tomioka: "Structural Analysis of Muscle Thin Filament" Advances in Biophysics. 27. (1990)
[Publications] Akihiro TOMIOKA: "Structure of Muscle Thin Filament in the Active State:Shape of “Polymeric"Actin" Journal of Molecular Biology.
[Publications] Ikawa,S.,Kamiya,N.,and Shibata,T.: "Defective Homologous Pairing and Proficient Processive Unwinding by the recA430 Mutant Protein and Intermediate ofHomologous Pairing by recA Protein." Journal of Biological Chemistry. 264. 21167-21176 (1989)
[Publications] A.Kusumi: "Liposomes that can be disintegrated by photoirradiation" Chem.Lett.433-436 (1989)
[Publications] W.K.Subczynski: "Oxygen permeability of phosphatidylcholine-cholesterol membranes" Proc.Natl.Acad.Sci.U.S.A.86. 4474-4478 (1989)
[Publications] M.Pasenkiewicz-Gierula: "Rotational diffusion of a steroid molecule in phosphatidylcholine cholesterol membranes:fluid phase micro-immiscibility in unsaturated-phosphatidylcholine-cholesterol membranes" Biochemistry. (1990)
[Publications] A.Kusumi: "Development of time-resolved microfluorimetry and its application to studies of cellular membranes" SPIE Proc.in press. 1204. (1990)
[Publications] C.J.Jones.H.Okino R.M.Macnab.S.Aizawa.: "Stoichiometnic Analysis of the Flagellar Hook-(Basal-Body)Complex of S.typhimurium" J.Mol.Biol.211. (1990)
[Publications] Y.Shirakihara: "Improved Preparations of Major Subunits of β and α of F1-ATPase from a Thermophilic Bacterium,and Spherulytes Formation in Crystallization Experiment Using Them" J.Biochem.
[Publications] J.Tokunaga,T.Iwasa,M.Takao,S.Sato,and T.Takeuchi: "Preparation and Properties of Monoclonal Antibodies against Bovine Rholopsin" Zoological Science. 6. 167-171 (1989)
[Publications] F.Tokunaga and T.Iwasa: "Human Color Pigment Gene" Seibutu-Buturi. 29. 311-315 (1989)
[Publications] Morikuni Yanagihara,Makiko Suwa and Shigeki Mitaku: "A Theoretical Method for Distinguishing between Soluble and Membrane Proteins" Biophys.Chem.34. 69-77 (1989)
[Publications] Shigeki Mitaku,Kazuya Ikuta,Hiroyasu Itoh Ryoichi Kataoda,Mami Naka,Naki Yamada and Makiko Suwa: "Denaturation of Bacteriorhodopsin by Organic Solvents" Biophys.Chem.30. 69-70 (1989)
[Publications] Tsuda,M.,Tsuda,T.Hirata,H.: "Cyclic mucleotides and GTP analoques stimutate light-induced phosphorylation of octopus rhodopsin" FEBS Letters. 257. 38-40 (1989)
[Publications] Bagley,K.Eisenstein,L Ebrey,T.Tsuda,M.: "A comparative Study of the Intrared difference Spectra for Octopus and bovine rhodopsin and their bathorhodopsin photointermediates" Biochemistry. 28. 3366-3373 (1989)
[Publications] Yoshikawa,S.,Shinzawa,K.,Tsukihara,T.,Abe,T.,W.S.Caughey.: "Crystallization of Beef Heart Cytochrome c Oxidase" J.Cryst.Growth.(1990)
[Publications] 吉川信也: "チトクロム酸化酵素の結晶化" 生物物理. 29. 256-259 (1989)
[Publications] 三木邦夫、J.Deisenhofer、H.Michel: "紅色光合成細菌の反応中心複合体の立体構造" 蛋白質・核酸・酵素. 34. 726-740 (1989)
[Publications] 三木邦夫、原田茂春: "蒸気拡散法によるタンパク質の結晶化-その特徴、結晶化用器具などについて" 日本結晶学会誌. 31. 256-260 (1989)
[Publications] 三木邦夫、佐野千明、三井幸雄(松崎通生、中島暉躬編): "新基礎生化学実験法、第5巻「高次構造・状態分析」(3.8節×線解析のための結晶化を分担執筆)" 丸善, 350 (1989)
[Publications] 三木邦夫(生物物理学会誌): "生命科学の基礎6「生体膜の分子素子・分子機械」(II・2節光合成膜の反応中心複合体)" 学会出版センタ-, (1990)
[Publications] 木下一彦、神山勉、池上明: "新基礎生化学実験法「高次構造・状態分析」-蛍光・蛍光偏光-" 丸善編者;中島暉躬、野本明男、松橋通生、三浦護一郎、村松正実, 16 (69-84) (1989)
[Publications] 神山勉、木下一彦: "分光技術ハンドブック「分光測定基礎技術」" 朝倉書店, 7

1989 Fiscal Year Annual Research Report

膜蛋白質の構造決定因子の解明と結晶化技術の開発

Principal Investigator

神山 勉 理化学研究所, 生物物理研究室, 研究員 (30170210)

Research Products

[Publications] Kouyama,T.,Kinosita,K.,Jr.,Ikegami,A.: "Correlation between internal motion and emission kinetics of tryptophan residues in proteins." Eur.J.Biochem.182. 517-521 (1989)

[Publications] Kouyama,T.,Nasuda-kouyama,A.: "Turnover rate of the proton pumping cycle of bacterorhodopsin:pH and light-intensity dependences." Biochemistry.28. 5963-5970 (1989)

[Publications] Fukuda,K.,Ikegami,A.,Nasuda-Kouyama,A.,Kouyama,T.: "Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin." Biochemistry.

[Publications] Nasuda-Kouyama,A.,Fukuda,K.,Iio,T.,Kouyama,T.: "Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin." Biochemistry.

[Publications] Akihiro Tomioka: "Structural Analysis of Muscle Thin Filament" Advances in Biophysics. 27. (1990)

[Publications] Akihiro TOMIOKA: "Structure of Muscle Thin Filament in the Active State:Shape of “Polymeric"Actin" Journal of Molecular Biology.

[Publications] Ikawa,S.,Kamiya,N.,and Shibata,T.: "Defective Homologous Pairing and Proficient Processive Unwinding by the recA430 Mutant Protein and Intermediate ofHomologous Pairing by recA Protein." Journal of Biological Chemistry. 264. 21167-21176 (1989)

[Publications] A.Kusumi: "Liposomes that can be disintegrated by photoirradiation" Chem.Lett.433-436 (1989)

[Publications] W.K.Subczynski: "Oxygen permeability of phosphatidylcholine-cholesterol membranes" Proc.Natl.Acad.Sci.U.S.A.86. 4474-4478 (1989)

[Publications] M.Pasenkiewicz-Gierula: "Rotational diffusion of a steroid molecule in phosphatidylcholine cholesterol membranes:fluid phase micro-immiscibility in unsaturated-phosphatidylcholine-cholesterol membranes" Biochemistry. (1990)

[Publications] A.Kusumi: "Development of time-resolved microfluorimetry and its application to studies of cellular membranes" SPIE Proc.in press. 1204. (1990)

[Publications] C.J.Jones.H.Okino R.M.Macnab.S.Aizawa.: "Stoichiometnic Analysis of the Flagellar Hook-(Basal-Body)Complex of S.typhimurium" J.Mol.Biol.211. (1990)

[Publications] Y.Shirakihara: "Improved Preparations of Major Subunits of β and α of F1-ATPase from a Thermophilic Bacterium,and Spherulytes Formation in Crystallization Experiment Using Them" J.Biochem.

[Publications] J.Tokunaga,T.Iwasa,M.Takao,S.Sato,and T.Takeuchi: "Preparation and Properties of Monoclonal Antibodies against Bovine Rholopsin" Zoological Science. 6. 167-171 (1989)

[Publications] F.Tokunaga and T.Iwasa: "Human Color Pigment Gene" Seibutu-Buturi. 29. 311-315 (1989)

[Publications] Morikuni Yanagihara,Makiko Suwa and Shigeki Mitaku: "A Theoretical Method for Distinguishing between Soluble and Membrane Proteins" Biophys.Chem.34. 69-77 (1989)

[Publications] Shigeki Mitaku,Kazuya Ikuta,Hiroyasu Itoh Ryoichi Kataoda,Mami Naka,Naki Yamada and Makiko Suwa: "Denaturation of Bacteriorhodopsin by Organic Solvents" Biophys.Chem.30. 69-70 (1989)

[Publications] Tsuda,M.,Tsuda,T.Hirata,H.: "Cyclic mucleotides and GTP analoques stimutate light-induced phosphorylation of octopus rhodopsin" FEBS Letters. 257. 38-40 (1989)

[Publications] Bagley,K.Eisenstein,L Ebrey,T.Tsuda,M.: "A comparative Study of the Intrared difference Spectra for Octopus and bovine rhodopsin and their bathorhodopsin photointermediates" Biochemistry. 28. 3366-3373 (1989)

[Publications] Yoshikawa,S.,Shinzawa,K.,Tsukihara,T.,Abe,T.,W.S.Caughey.: "Crystallization of Beef Heart Cytochrome c Oxidase" J.Cryst.Growth.(1990)

[Publications] 吉川信也: "チトクロム酸化酵素の結晶化" 生物物理. 29. 256-259 (1989)

[Publications] 三木邦夫、J.Deisenhofer、H.Michel: "紅色光合成細菌の反応中心複合体の立体構造" 蛋白質・核酸・酵素. 34. 726-740 (1989)

[Publications] 三木邦夫、原田茂春: "蒸気拡散法によるタンパク質の結晶化-その特徴、結晶化用器具などについて" 日本結晶学会誌. 31. 256-260 (1989)

[Publications] 三木邦夫、佐野千明、三井幸雄(松崎通生、中島暉躬編): "新基礎生化学実験法、第5巻「高次構造・状態分析」(3.8節×線解析のための結晶化を分担執筆)" 丸善, 350 (1989)

[Publications] 三木邦夫(生物物理学会誌): "生命科学の基礎6「生体膜の分子素子・分子機械」(II・2節 光合成膜の反応中心複合体)" 学会出版センタ-, (1990)

[Publications] 木下一彦、神山勉、池上明: "新基礎生化学実験法「高次構造・状態分析」-蛍光・蛍光偏光-" 丸善 編者;中島暉躬、野本明男、松橋通生、三浦護一郎、村松正実, 16 (69-84) (1989)

[Publications] 神山勉、木下一彦: "分光技術ハンドブック「分光測定基礎技術」" 朝倉書店, 7

神山勉理化学研究所, 生物物理研究室, 研究員 (30170210)

[Publications] 三木邦夫(生物物理学会誌): "生命科学の基礎6「生体膜の分子素子・分子機械」(II・2節光合成膜の反応中心複合体)" 学会出版センタ-, (1990)

[Publications] 木下一彦、神山勉、池上明: "新基礎生化学実験法「高次構造・状態分析」-蛍光・蛍光偏光-" 丸善編者;中島暉躬、野本明男、松橋通生、三浦護一郎、村松正実, 16 (69-84) (1989)