1996 年度実績報告書

シグナル伝達系におけるレセプターG蛋白質相互作用の原子レベルでの解析

研究課題

研究課題/領域番号	08308038
研究種目	基盤研究(A)
応募区分	総合
研究機関	京都大学
研究代表者	七田芳則京都大学, 大学院・理学研究科, 助教授 (60127090)
研究分担者	藤吉芳則京都大学, 大学院・理学研究科, 教授 (80142298) 徳永史生大阪大学, 大学院・理学研究科, 教授 (80025452) 津田基之姫路工業大学, 理学部, 教授 (60045458) 加茂直樹北海道大学, 薬学部, 教授 (10001976) 垣谷俊昭名古屋大学, 大学院・理学研究科, 教授 (90027350)
キーワード	受容体 / G蛋白質 / ロドプシン / バクテリオロドプシン / 光 / 情報 / 構造 / 機能
研究概要	多くの生体内情報伝達系の中核を担う受容体-G蛋白質の相互作用機構を解明するために、視覚系の光受容体であるロドプシンと、G蛋白質トランスデューシンの相互作用機構を生物物理学的、生化学的手法により、検討した。その結果、下記の成果が得られた。 (1)ロドプシンなどの光受容蛋白質内部における光情報伝達機構・膜貫通領域に存在するアミノ酸残基のうち、桿体及び錐体の光情報伝達効率を制御するアミノ酸残基を同定した。・バクテリオロドプシン(bR)を偏光で励起し、異方性を経時的に測定した結果、bRが励起される分子運動が遅くなることがわかった. ・C_<13>=C_<14>を固定した発色団レチナ-ルを組み込んだバクテリオロドプシンの励起状態ダイナミックスを解析した結果、C_<13>=C_<14>のねじれる動きは100fs過ぎてから本格的に進行する事がわかった。 (2)ロドプシンとG蛋白質の相互作用脊椎動物視細胞ではロドプシンの中間体メタロドプシンIIが、G蛋白質を活性化する。・メタII生成時のFTIRスペクトルを測定した結果、G蛋白質と結合したときに蛋白質で生じる構造変化を分子振動のレベルで検出することができた。・無脊椎動物では、メタロドプシンでなくその前駆体がG蛋白質を活性化することを見いだした。 (3)細胞内光情報伝達機構・桿体のトランスデューシンは、4種類の脂肪酸によって不均一に修飾されているが、その局在を検討した結果、4種類のT_αは一つの桿体視細胞に共存していると推定された。・桿体の信号伝達についてモデルを用いて検討した結果、通常の拡散を基本にした信号伝達メカニズムでは、測定されている膜電流変化の速い時間経過を説明できない可能性を示した。 (4)受容体-G蛋白質結合構造の解明・ウシ桿体細胞外節膜の可溶化過程を検討し、ロドプシンのみを選択的に得る条件を決めた。その試料を用いて、2種類以上の赤い3次元単結晶が得られた。

研究成果
(56件)

すべてその他

すべて文献書誌 (56件)

[文献書誌] D.Kojima: "Molecular properies of chimerical mutants of gecko blue and bovine rhodopsin." Biochemistry. 35. 2625-2629 (1996)
[文献書誌] H.Kandori: "Real-time detection of 60-femtosecond isomerization in a rhodopsin analog containing 8-membered-ring-retinal." J.Am.Chem.Soc.118. 1002-1005 (1996)
[文献書誌] Y.Imamoto: "Structure around C6-C7 bond of the chromophore in bathorhodopsin : Low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs" Biochemistry. 35. 6257-6262 (1996)
[文献書誌] Y.Imamoto: "Chromophore configuration of iodopsin and its photoproducts formed at low temperatures." Biochemistry. 35. 14599-14607 (1996)
[文献書誌] H.Imai: "Single Amino Acid Residue as a Functional Determinant of Rod and Cone Visual Pigments." Proc.Nalt.Acad.Sci.USA. (印刷中).
[文献書誌] R.Akiyama: "Analysis of temperature dependence of femsecond excited state synamics of bacteriorhodopsin by spin-boson model." Chem.Phys.Lett.256. 165-171 (1996)
[文献書誌] R.Akiyama: "Analtsis of the Excited-State dynamics of 13-trans-locked-bacteriorhodopsin." J.Phys.Chem.101. 412-417 (1997)
[文献書誌] T.Kitajima: "Novel bacterial rhodopsins from Haloarcula vallismortis." Biochem.Biophys.Res.Commun.220. 341-345 (1996)
[文献書誌] T.Kikukawa: "Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane." Chem.Pharm.Bull.44. 473-476 (1996)
[文献書誌] T.Kikukawa: "Restricted motion of photo-excited bacteriorhodopsin in purple membranes containing ethanol." Biophys.J.(in press). (1997)
[文献書誌] S.Kikkawa: "Simple Purification and Functional Reconstitution of Octopus Photoreceptor Gq,Which Couples Rhodospin to Phospholipase C" Biochemistry. 35. 15857-15864 (1996)
[文献書誌] S.Hashimoto: "Ultraviolet Resonance Raman Evidence for the Absence of Tyrosine in Octopus Rhodopsin and the Participation of Trp residues in the Transition to Acid Metarhodopsin." FEBS Lett.398. 239-242 (1996)
[文献書誌] L.Huang: "A Resonance Raman Study of the C=N Configuration of Octopus Rhodopsin Bathorhodopsin,and Isorhodopsin" Biochemistry. 35. 8505-8510 (1996)
[文献書誌] M.Nakagawa: "Identification of Two Palmitoyl Groups in Octopus Rhodopsin." Photochem.Photobiol.65. 187-191 (1997)
[文献書誌] S.Nishimura: "Dynamics of Waters and the Peptide Backbone around the Schiff Base Associated with the Light-ativated Process of Octopus Rhodopsin." Biochemistry. 36. 864-870 (1997)
[文献書誌] M.Nakagawa: "Light-induced Protein Conformational Changes in the Photolysis of Octopus Rhodopsin." Biophys.J.in press. (1997)
[文献書誌] Y.Tsujiuchi: "Expression of partial genes of bacteriohordopsin in Escherichia coli" Interdiscipl.Inform.Sci.2. 39-45 (1996)
[文献書誌] K.Sakamoto: "Two opsins from the compound eye of the crab,Hemigrapsus sanguineus" J.Exp.Biol.199. 441-450 (1996)
[文献書誌] O.Hisatomi: "Isolation and characterization of the putative goldfish UV-sensitive pigment cDNA" Vision Res.35. 933-939 (1996)
[文献書誌] D.L.Stemkamp: "Temporal expression of rod and cone opsins in embryonic goldfish redina predicts the spacial organization to the cone mosaic." Invest.Ophthal.Vis.Sci.37. 363-376 (1996)
[文献書誌] S.Kawamura: "Photoreceptor protein s26,a cone homologue of S-modulin in frog retina" J.Biol.Chem.271. 21359-21364 (1996)
[文献書誌] M.Ishikawa: "Lectin cytochemical analysis of glycoconjugates in photoreceptor cell membranes of Lampetra" Vision Res.36. 1513-1520 (1996)
[文献書誌] A.Hikikoshi-Iwane: "Two-dimensional crystals of the Kdp-ATPase of escherichia coli." FEBS Letters. 396. 172-176 (1996)
[文献書誌] Y.Mimori-Kiyosue: "Direct interaction of flagellin termini essential for polymorphic ability of flagellar filament." Proc.natle.Acad.Sci.USA. 93. 15108-15113 (1996)
[文献書誌] K.Mitsuoka: "Examination of the leafScan 45,a line-illuminating micro-densitometer,for its use in electron crystallography" Ultramicroscopy.(in press).
[文献書誌] T.Walz.: "Three-dimentional structure of Aquaporin 1 at 6 A." Nature. (in press).
[文献書誌] 村田和義: "蛋白質の高分解能構造解析のための試料作製法" 電子顕微鏡. 31 No.2/3. 149-152 (1996)
[文献書誌] 藤吉好則: "電子顕微鏡による生体高分子の立体構造解析" 生物物理. 207. 221-225 (1996)
[文献書誌] O.Shouno: "Preparation and characterization of monoclonal antibodies specific for lauroylated isoform of bovine transducin alpha-subunit : Immunohistochemical analysis of bovine retinas." J.Neurochem.(in press).
[文献書誌] K.Sanada: "Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation." FEBS Lett.384. 227-230 (1996)
[文献書誌] T.Okano: "Molecular cloning of heterotrimeric G-protein a-subunits in chicken pineal gland." J.Mol.Evol.(in press). (1997)
[文献書誌] Y.-S.Chon: "Hydration of the counterion of the Schiff base in the chloride transporting mutant of bacteriorhodopsin : FTIR and FT-Raman studies on the effects of anion binding when Asp85 is replaced with a neutral residue" Biochemistry. 35. 14244-14250 (1996)
[文献書誌] Y.Yamazaki: "Hydrogen-bonds of water and C=O groups coordinate long-range structural changes in the L photointermediate of bacteriorhodopsin." Biochemistry. 35. 4063-4068 (1996)
[文献書誌] M.Hatanaka: "Effects of arginine-82 on the interactions of internal water molecules in bacteriorhodopsin." Biochemistry. 35. 6308-6312 (1996)
[文献書誌] H.Richter: "Relationship of retinal configuration and internal proton transfer at the end of the bacteriorhodopsin photocycle." Biochemistry. 35. 15461-15466 (1996)
[文献書誌] S.Nishimura: "Structural changes in the reptide backbone in complex formation between acivated rhodopsin and transducin studied by FTIR spectroscopy." Biochemistry. 35. 13267-13271 (1996)
[文献書誌] A.Maeda: "Intramembrane Signaling Mediated by Hydrogen-Bonding of Water and Carboxyl Groups in Bacteriorphodopsin and Rhodopsin." J.Biochem.121. 399-406 (1997)
[文献書誌] A.Maeda: "Application of FT-IR spectroscopy to the structural study on the function of bacteriorhodopsin." Israel J.Chem.35. 387-400 (1996)
[文献書誌] 神取秀樹: "バクテリオロドプシンとハロロドプシン" 化学. 51. 32-37 (1996)
[文献書誌] 西村章子: "ロドプシンの活性化反応と水分子" Molecular Medicine. 33. 718-719 (1996)
[文献書誌] 神取秀樹: "バクテリオロドプシンはどのようにして光をエネルギーに変換するのか?" 蛋白質・核酸・酵素. 42. 101-109 (1997)
[文献書誌] K.Ichikawa: "Modeling and analysis of spatio-temporal change in [Ca2+]i in a retinal rod outer segment" Neurosci.Res.25. 137-144 (1996)
[文献書誌] T.Matsuhashi: "Mechanism of formation of megamitochondria in the mouse liver induced chloramphenicol." Toxicol.Lett.86. 47-54 (1996)
[文献書誌] Y.Hirako: "Demonstration of the molecular shape of BP 180,a 180 kDa bullous pemphigoid antigen,and its potential for trimer formation." J.Biol.Chem.271. 13739-13745 (1996)
[文献書誌] S.Taya: "Development of g type energy filtering TEM." J.Electron Microsc.45. 307-313 (1996)
[文献書誌] A.Yamazaki: "Possible stimulation of retinal rod recovery to dark state by cGMP release from a cGMP phosphodiesterase noncatalytic site." J.Biol.Chem.271. 32495-32498 (1996)
[文献書誌] 岡田哲二: "膜蛋白質の構造と機能" 膜. 21. 230-239 (1997)
[文献書誌] H.Itoh: "Estimation of the Hydrophobicity in Microenvironments by Pyren Fluorescence Mesurements : n-β-Octylglucoside Micelles." J.Phys.Chem.100. 9047-9053 (1996)
[文献書誌] A.Etoh: "Light-Induced Denaturation of Bacteriorhodopsin Just Above Melting Point of Two-Dimensional Crystal." Journal of The Physical Society of Japan. (印刷中).
[文献書誌] A.Etoh: "Anomalous Ultrasonic Absorption of Liposome Suspensions in the Liquid Crystal Phase." Japanese Journal of Applied Physics. (印刷中).
[文献書誌] 七田芳則: "生物物理から見た生命像4「感情情報-生物の巧みなシグナルのやり取り」(分担執筆)" 吉岡書店(印刷中),
[文献書誌] 津田基之: "タンパク質超分子システムの構造と機能「新タンパク質応用工学」" フィジテクノシステム, 6 (1996)
[文献書誌] 津田基之: "信号伝達,生物物理から見た生命像2「生体膜-生命の基本形をかたちづくるもの」" 吉岡書店, 13 (1996)
[文献書誌] 津田基之: "パリティ「ものが見えるとはどういうことか視覚の初期過程の分子メカニズム」" 丸善, 8 (1996)
[文献書誌] 美宅成樹: "シリーズ・ニューバイオフィジックス (11)ヒューマンゲノム計画第2章膜タンパクの分類" 共立出版(印刷中),
[文献書誌] 美宅成樹(訳): "Robert A.Day(著)、はじめての科学英語論文" 丸善, 293 (1997)

1996 年度 実績報告書

シグナル伝達系におけるレセプターG蛋白質相互作用の原子レベルでの解析

研究代表者

七田 芳則 京都大学, 大学院・理学研究科, 助教授 (60127090)

研究成果

[文献書誌] D.Kojima: "Molecular properies of chimerical mutants of gecko blue and bovine rhodopsin." Biochemistry. 35. 2625-2629 (1996)

[文献書誌] H.Kandori: "Real-time detection of 60-femtosecond isomerization in a rhodopsin analog containing 8-membered-ring-retinal." J.Am.Chem.Soc.118. 1002-1005 (1996)

[文献書誌] Y.Imamoto: "Structure around C6-C7 bond of the chromophore in bathorhodopsin : Low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs" Biochemistry. 35. 6257-6262 (1996)

[文献書誌] Y.Imamoto: "Chromophore configuration of iodopsin and its photoproducts formed at low temperatures." Biochemistry. 35. 14599-14607 (1996)

[文献書誌] H.Imai: "Single Amino Acid Residue as a Functional Determinant of Rod and Cone Visual Pigments." Proc.Nalt.Acad.Sci.USA. (印刷中).

[文献書誌] R.Akiyama: "Analysis of temperature dependence of femsecond excited state synamics of bacteriorhodopsin by spin-boson model." Chem.Phys.Lett.256. 165-171 (1996)

[文献書誌] R.Akiyama: "Analtsis of the Excited-State dynamics of 13-trans-locked-bacteriorhodopsin." J.Phys.Chem.101. 412-417 (1997)

[文献書誌] T.Kitajima: "Novel bacterial rhodopsins from Haloarcula vallismortis." Biochem.Biophys.Res.Commun.220. 341-345 (1996)

[文献書誌] T.Kikukawa: "Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane." Chem.Pharm.Bull.44. 473-476 (1996)

[文献書誌] T.Kikukawa: "Restricted motion of photo-excited bacteriorhodopsin in purple membranes containing ethanol." Biophys.J.(in press). (1997)

[文献書誌] S.Kikkawa: "Simple Purification and Functional Reconstitution of Octopus Photoreceptor Gq,Which Couples Rhodospin to Phospholipase C" Biochemistry. 35. 15857-15864 (1996)

[文献書誌] S.Hashimoto: "Ultraviolet Resonance Raman Evidence for the Absence of Tyrosine in Octopus Rhodopsin and the Participation of Trp residues in the Transition to Acid Metarhodopsin." FEBS Lett.398. 239-242 (1996)

[文献書誌] L.Huang: "A Resonance Raman Study of the C=N Configuration of Octopus Rhodopsin Bathorhodopsin,and Isorhodopsin" Biochemistry. 35. 8505-8510 (1996)

[文献書誌] M.Nakagawa: "Identification of Two Palmitoyl Groups in Octopus Rhodopsin." Photochem.Photobiol.65. 187-191 (1997)

[文献書誌] S.Nishimura: "Dynamics of Waters and the Peptide Backbone around the Schiff Base Associated with the Light-ativated Process of Octopus Rhodopsin." Biochemistry. 36. 864-870 (1997)

[文献書誌] M.Nakagawa: "Light-induced Protein Conformational Changes in the Photolysis of Octopus Rhodopsin." Biophys.J.in press. (1997)

[文献書誌] Y.Tsujiuchi: "Expression of partial genes of bacteriohordopsin in Escherichia coli" Interdiscipl.Inform.Sci.2. 39-45 (1996)

[文献書誌] K.Sakamoto: "Two opsins from the compound eye of the crab,Hemigrapsus sanguineus" J.Exp.Biol.199. 441-450 (1996)

[文献書誌] O.Hisatomi: "Isolation and characterization of the putative goldfish UV-sensitive pigment cDNA" Vision Res.35. 933-939 (1996)

[文献書誌] D.L.Stemkamp: "Temporal expression of rod and cone opsins in embryonic goldfish redina predicts the spacial organization to the cone mosaic." Invest.Ophthal.Vis.Sci.37. 363-376 (1996)

[文献書誌] S.Kawamura: "Photoreceptor protein s26,a cone homologue of S-modulin in frog retina" J.Biol.Chem.271. 21359-21364 (1996)

[文献書誌] M.Ishikawa: "Lectin cytochemical analysis of glycoconjugates in photoreceptor cell membranes of Lampetra" Vision Res.36. 1513-1520 (1996)

[文献書誌] A.Hikikoshi-Iwane: "Two-dimensional crystals of the Kdp-ATPase of escherichia coli." FEBS Letters. 396. 172-176 (1996)

[文献書誌] Y.Mimori-Kiyosue: "Direct interaction of flagellin termini essential for polymorphic ability of flagellar filament." Proc.natle.Acad.Sci.USA. 93. 15108-15113 (1996)

[文献書誌] K.Mitsuoka: "Examination of the leafScan 45,a line-illuminating micro-densitometer,for its use in electron crystallography" Ultramicroscopy.(in press).

[文献書誌] T.Walz.: "Three-dimentional structure of Aquaporin 1 at 6 A." Nature. (in press).

[文献書誌] 村田和義: "蛋白質の高分解能構造解析のための試料作製法" 電子顕微鏡. 31 No.2/3. 149-152 (1996)

[文献書誌] 藤吉 好則: "電子顕微鏡による生体高分子の立体構造解析" 生物物理. 207. 221-225 (1996)

[文献書誌] O.Shouno: "Preparation and characterization of monoclonal antibodies specific for lauroylated isoform of bovine transducin alpha-subunit : Immunohistochemical analysis of bovine retinas." J.Neurochem.(in press).

[文献書誌] K.Sanada: "Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation." FEBS Lett.384. 227-230 (1996)

[文献書誌] T.Okano: "Molecular cloning of heterotrimeric G-protein a-subunits in chicken pineal gland." J.Mol.Evol.(in press). (1997)

[文献書誌] Y.-S.Chon: "Hydration of the counterion of the Schiff base in the chloride transporting mutant of bacteriorhodopsin : FTIR and FT-Raman studies on the effects of anion binding when Asp85 is replaced with a neutral residue" Biochemistry. 35. 14244-14250 (1996)

[文献書誌] Y.Yamazaki: "Hydrogen-bonds of water and C=O groups coordinate long-range structural changes in the L photointermediate of bacteriorhodopsin." Biochemistry. 35. 4063-4068 (1996)

[文献書誌] M.Hatanaka: "Effects of arginine-82 on the interactions of internal water molecules in bacteriorhodopsin." Biochemistry. 35. 6308-6312 (1996)

[文献書誌] H.Richter: "Relationship of retinal configuration and internal proton transfer at the end of the bacteriorhodopsin photocycle." Biochemistry. 35. 15461-15466 (1996)

[文献書誌] S.Nishimura: "Structural changes in the reptide backbone in complex formation between acivated rhodopsin and transducin studied by FTIR spectroscopy." Biochemistry. 35. 13267-13271 (1996)

[文献書誌] A.Maeda: "Intramembrane Signaling Mediated by Hydrogen-Bonding of Water and Carboxyl Groups in Bacteriorphodopsin and Rhodopsin." J.Biochem.121. 399-406 (1997)

[文献書誌] A.Maeda: "Application of FT-IR spectroscopy to the structural study on the function of bacteriorhodopsin." Israel J.Chem.35. 387-400 (1996)

[文献書誌] 神取秀樹: "バクテリオロドプシンとハロロドプシン" 化学. 51. 32-37 (1996)

[文献書誌] 西村章子: "ロドプシンの活性化反応と水分子" Molecular Medicine. 33. 718-719 (1996)

[文献書誌] 神取秀樹: "バクテリオロドプシンはどのようにして光をエネルギーに変換するのか?" 蛋白質・核酸・酵素. 42. 101-109 (1997)

[文献書誌] K.Ichikawa: "Modeling and analysis of spatio-temporal change in [Ca2+]i in a retinal rod outer segment" Neurosci.Res.25. 137-144 (1996)

[文献書誌] T.Matsuhashi: "Mechanism of formation of megamitochondria in the mouse liver induced chloramphenicol." Toxicol.Lett.86. 47-54 (1996)

[文献書誌] Y.Hirako: "Demonstration of the molecular shape of BP 180,a 180 kDa bullous pemphigoid antigen,and its potential for trimer formation." J.Biol.Chem.271. 13739-13745 (1996)

[文献書誌] S.Taya: "Development of g type energy filtering TEM." J.Electron Microsc.45. 307-313 (1996)

[文献書誌] A.Yamazaki: "Possible stimulation of retinal rod recovery to dark state by cGMP release from a cGMP phosphodiesterase noncatalytic site." J.Biol.Chem.271. 32495-32498 (1996)

[文献書誌] 岡田哲二: "膜蛋白質の構造と機能" 膜. 21. 230-239 (1997)

[文献書誌] H.Itoh: "Estimation of the Hydrophobicity in Microenvironments by Pyren Fluorescence Mesurements : n-β-Octylglucoside Micelles." J.Phys.Chem.100. 9047-9053 (1996)

[文献書誌] A.Etoh: "Light-Induced Denaturation of Bacteriorhodopsin Just Above Melting Point of Two-Dimensional Crystal." Journal of The Physical Society of Japan. (印刷中).

[文献書誌] A.Etoh: "Anomalous Ultrasonic Absorption of Liposome Suspensions in the Liquid Crystal Phase." Japanese Journal of Applied Physics. (印刷中).

[文献書誌] 七田芳則: "生物物理から見た生命像4「感情情報-生物の巧みなシグナルのやり取り」(分担執筆)" 吉岡書店(印刷中),

[文献書誌] 津田基之: "タンパク質超分子システムの構造と機能「新タンパク質応用工学」" フィジテクノシステム, 6 (1996)

[文献書誌] 津田基之: "信号伝達,生物物理から見た生命像2「生体膜-生命の基本形をかたちづくるもの」" 吉岡書店, 13 (1996)

[文献書誌] 津田基之: "パリティ「ものが見えるとはどういうことか 視覚の初期過程の分子メカニズム」" 丸善, 8 (1996)

[文献書誌] 美宅成樹: "シリーズ・ニューバイオフィジックス (11)ヒューマンゲノム計画 第2章 膜タンパクの分類" 共立出版(印刷中),

[文献書誌] 美宅成樹(訳): "Robert A.Day(著)、はじめての科学英語論文" 丸善, 293 (1997)

1996 年度実績報告書

七田芳則京都大学, 大学院・理学研究科, 助教授 (60127090)

[文献書誌] 藤吉好則: "電子顕微鏡による生体高分子の立体構造解析" 生物物理. 207. 221-225 (1996)

[文献書誌] 津田基之: "パリティ「ものが見えるとはどういうことか視覚の初期過程の分子メカニズム」" 丸善, 8 (1996)

[文献書誌] 美宅成樹: "シリーズ・ニューバイオフィジックス (11)ヒューマンゲノム計画第2章膜タンパクの分類" 共立出版(印刷中),