シャペロニン機能の1分子イメージング

• Project/Area Number: 06J10141
• Research Category: Grant-in-Aid for JSPS Fellows
• Allocation Type: Single-year Grants
• Section: 国内
• Research Field: Biophysics
• Research Institution: The University of Tokyo
• Principal Investigator: 飯塚 怜 The University of Tokyo, 大学院・薬学系研究科, 特別研究員(PD)
• Project Period (FY): 2006 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2008: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2007: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2006: ¥1,200,000 (Direct Cost: ¥1,200,000)
• Keywords: シャペロニン / 構造変化 / 1分子蛍光イメージング / 古細菌

Research Abstract

バクテリア型及び真核細胞型シャペロニンが「どのようにタンパク質のフォールディングを介助しているか」を明らかにすることを目指し、下記のような研究を遂行した。
1.バクテリア型シャペロニンの分子機構解析
大腸菌のシャペロニンGroELが生理的条件下において、弾丸型複合体およびフットボール型複合体を同程度の割合で形成することを明らかにした。今日まで、GroELの機能は弾丸型複合体を通じて発揮されるとされてきた。今回の発見はこの定説を覆すものであり、GroELの機能発現機構モデルの改訂を迫る発見と言える。この成果はJournal of Biological Chemistry誌に掲載された。
2.真核細胞型シャペロニンの分子機構解析
超好熱性古細菌Thermococcus sp.strain KS-1(T.KS-1)由来シャペロニンの基質タンパク質を取り込む空洞部分の蓋構造(Helical protrusion)の開閉を(1)生化学的・構造学的に(2)1分子レベルで解析した。(1)T.KS-1シャペロニンサブユニットの連結変異体を用いた実験から、ATPにより各サブユニットで構造変化が起こった後、隣接したサブユニットのHelical protrusion間で相互作用することで空洞部分が閉じた構造へと転換することを見出した。この成果は、Journal of Biological Chemistry誌に発表した。(2)Helical protrusionの開閉を、結合させた蛍光色素の配向を検出することで可視化した。蛍光色素の配向は、励起に直線偏光を用い、その偏光を回転させ、蛍光強度の経時変化から決定した。この実験系において構造変化の観察事例を増やすことに成功し、蛍光色素の配向の差異から見積もられる角度変化は過去の研究結果と一致した。現在、この成果の論文発表の準備を進めている。

Research Products

• [Journal Article] Two small heat shock proteins of a fission yeast, Schizosaccharomyces pombe, function in different manner to cope with wide range of temperatures and various denatured proteins (2009)
  • Author(s): Chika Sugino
  • Journal Title: Proteins : Structure, Function, and Bioinformatics 74 Pages: 6-17
  • Peer Reviewed
• [Journal Article] Single molecule imaging of full protein synthesis by immobilized ribosomes (2008)
  • Author(s): Sotaro Uemura
  • Journal Title: Nucleic Acids Research 36
  • Peer Reviewed
• [Journal Article] Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle and their number ratio is determined by nucleotide in the trans-ring (2008)
  • Author(s): Tomoya Sameshima
  • Journal Title: Journal of Biological Chemistry 283 Pages: 23765-23773
  • Peer Reviewed
• [Journal Article] Effect of the C-terminal truncation on the functional cycle of chaperonin GroEL : implication that the C-terminal region facilitates the transition from the folding-arrested to folding-competent state (2008)
  • Author(s): Mihoko Suzuki
  • Journal Title: Journal of Biological Chemistry 283 Pages: 23931-23939
  • Peer Reviewed
• [Journal Article] Structural and molecular characterization of the prefoldin β subunit from Thermococcus strain KS-1 (2008)
  • Author(s): Hiroshi Kida
  • Journal Title: Journal of Molecular Biology 383 Pages: 465-474
  • Peer Reviewed
• [Journal Article] Interaction between helical protrusions induces closure of the built-in lid of group II chaperonins (2008)
  • Author(s): Taro Kanzaki
  • Journal Title: Journal of Biological Chemistry 283 Pages: 34773-34784
  • Peer Reviewed
• [Journal Article] (2008)
  • Author(s): Tamotsu Zako
  • Journal Title: Heat-Shock Proteins : International Research(Nova Science Publishers) Pages: 393-416
• [Journal Article] Structure of aspartate racemase complexed with a dual substrate analogue,citricacid,and implications for the reaction mechanism (2008)
  • Author(s): Akashi Ohtaki
  • Journal Title: Proteins:Structure, Function, and Bioinformatics 70 Pages: 1167-1174
  • Peer Reviewed
• [Journal Article] Dynamics of group II chaperonin and prefoldin probed by ^<13>C NMR spectroscopy (2008)
  • Author(s): Eiji Kurimoto
  • Journal Title: Proteins:Structure, Function, and Bioinformatics 70 Pages: 1257-1263
  • Peer Reviewed
• [Journal Article] Structure and Molecular Dynamics Simulation of Archaeal Prefoldin:The Molecular Mechanism for Binding and Recognition of Nonnative Substrate Proteins (2008)
  • Author(s): Akashi Ohtaki
  • Journal Title: Journal of Molecular Biology 376 Pages: 771-782
  • Peer Reviewed
• [Journal Article] Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon,Sulfolobus tokodaii strain 7 (2008)
  • Author(s): Hitoshi Saji
  • Journal Title: Proteins:Structure, Function, and Bioinformatics 71 Pages: 1130-1141
  • Peer Reviewed
• [Journal Article] Functional characterization of prefoldin complexes from a hyperthermophilic archaeon,Thermococcus sp.stain KS-1 (2008)
  • Author(s): Ryo Iizuka
  • Journal Title: Journal of Molecular Biology 377 Pages: 972-983
  • Peer Reviewed
• [Journal Article] Complex formation of CdSe/ZnS/TOPO nanocrystal vs. molecular chaperone in a queous solution by hydrophobic interaction (2007)
  • Author(s): Hiromi Horiuchi
  • Journal Title: Journal of Luminescence 127 Pages: 192-197
  • Peer Reviewed
• [Journal Article] Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1 : the effects of beryllium fluoride. (2007)
  • Author(s): Takao Yoshida
  • Journal Title: Extremophiles 11(2) Pages: 225-235
• [Journal Article] Contribution of the C-terminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS-1. (2006)
  • Author(s): Takao Yoshida
  • Journal Title: Extremophiles 10(5) Pages: 451-459
• [Journal Article] Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlation between binding rate and protein transfer rate. (2006)
  • Author(s): Tamotsu Zako
  • Journal Title: Journal of Molecular Biology 364(1) Pages: 110-120
• [Presentation] Thermococcus strain KS-1由来プレフォルディンβサブユニットの結晶構造 (2009)
  • Author(s): 木田宗志
  • Organizer: 第26回PFシンポジウム
  • Place of Presentation: つくば国際会議場(茨城)
  • Year and Date: 2009-03-25
• [Presentation] Interaction between Molecular Chaperone Prefoldin with Group II Chaperonin in the Presence of Nucleotides : Implication for Substrate Transfer Mechanism from Prefoldin to Chaperonin (2009)
  • Author(s): Tamotsu Zako
  • Organizer: Biophysical Society 53^<rd> Annual Meeting
  • Place of Presentation: Boston, CA
  • Year and Date: 2009-03-03
• [Presentation] GroEL-GroES相互作用に与える変性タンパク質の影響 (2008)
  • Author(s): 鮫島知哉
  • Organizer: 第31回日本分子生物学会年会・第81回日本生化学会年会合同大全BMB2008
  • Place of Presentation: 神戸ポートアイランド(兵庫)
  • Year and Date: 2008-12-09
• [Presentation] 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析 (2008)
  • Author(s): Ryo Iizuka
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡国際会議場(福岡)
  • Year and Date: 2008-12-04
• [Presentation] ナノ開口基板上でのタンパク質翻訳中における高濃度tRNAの1分子ダイナミクス (2008)
  • Author(s): Sotaro Uemura
  • Organizer: 日本生物物理学会第46回年会
  • Place of Presentation: 福岡国際会議場(福岡)
  • Year and Date: 2008-12-04
• [Presentation] 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析 (2008)
  • Author(s): 飯塚怜
  • Organizer: 文部科学省科学研究費補助金特定領域研究「タンパク質の社会」2008年度班会議
  • Place of Presentation: 残波岬ロイヤルホテル(沖縄)
  • Year and Date: 2008-11-24
• [Presentation] 真核細胞型シャペロニンによるタンパク質フォールディング機構 (2008)
  • Author(s): 飯塚怜
  • Organizer: 極限環境微生物学会第9回年会
  • Place of Presentation: 立教大学(東京)
  • Year and Date: 2008-11-05
• [Presentation] 1分子蛍光偏光法による古細菌型シャペロニンの構造変化解析 (2008)
  • Author(s): 飯塚怜
  • Organizer: 文部科学省科学研究費補助金特定領域研究「タンパク質の社会」若手ワークショップ
  • Place of Presentation: 東京大学検見川セミナーハウス(千葉)
  • Year and Date: 2008-09-26
• [Presentation] ATP-DEPENDENT CONFORMATIONAL CHANGE MECHANISM OF GROUP II CHAPERONINS ELUCIDATED USING CHAPERONIN COMPLEXES CONTAINING MUTANT SUBUNITS IN THE ORDERDED FASHION (2008)
  • Author(s): Masafumi Yohda
  • Organizer: 22^<nd> Annual Symposium of The Protein Society
  • Place of Presentation: San Diego, CA
  • Year and Date: 2008-07-22
• [Presentation] Prefoldin prudentially interacts with group II chaperonin in its ADP bound open conformation (2008)
  • Author(s): Tamotsu Zako
  • Organizer: 33^<rd> FEBS Congress & 11^<th> IUBMB Conference
  • Place of Presentation: Athens, Greece
  • Year and Date: 2008-06-30
• [Presentation] 古細菌型シャペロニンの1分子構造変化計測 (2008)
  • Author(s): 飯塚怜
  • Organizer: 第8回日本蛋白質科学会年会
  • Place of Presentation: タワーホール船堀(東京)
  • Year and Date: 2008-06-11
• [Presentation] Conjugated chaperonin を用いたグループII型シャペロニンのATP依存的構造変化機構の解析 (2008)
  • Author(s): 神前太郎
  • Organizer: 第8回日本蛋白質科学会年会
  • Place of Presentation: タワーホール船堀(東京)
  • Year and Date: 2008-06-11
• [Presentation] 超好熱性古細菌 Thermococcus sp. strain KS-1由来プレフォルディンの機能解析 (2008)
  • Author(s): 米澤哲洋
  • Organizer: 第8回日本蛋白質科学会年会
  • Place of Presentation: タワーホール船堀(東京)
  • Year and Date: 2008-06-11
• [Presentation] ATP-DEPENDENT CONFORMATIONAL CHANGE MECHANISM OF GROUP II CHAPERONINS ELUCIDATED USING CHAPERONIN COMPLEXES CONTAINING MUTANT SUBUNITS IN THE ORDERDED FASHION (2008)
  • Author(s): Taro Kanzaki
  • Organizer: Cold Spring Harbor Laboratory Meeting on Molecular chaperones and the Heat Shock Response
  • Place of Presentation: Cold Spring Harbor, NY
  • Year and Date: 2008-05-02
• [Presentation] Single Molecule Imaging Of Full Protein Synthesis Reconstitution By lmmobilized Ribosome (2008)
  • Author(s): Sotaro Uemura
  • Organizer: Joint Meeting of the Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress
  • Place of Presentation: Long Beach,CA
  • Year and Date: 2008-02-06
• [Presentation] ADP Regulates Interaction between Escherichia Coli Chaperonin GroEL and Its Cofactor GroES (2008)
  • Author(s): Tomoya Sameshima
  • Organizer: Joint Meeting of the Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress
  • Place of Presentation: Long Beach,CA
  • Year and Date: 2008-02-04
• [Presentation] Molecular mechanism and function of sHsps from the fission yeast,Schizosaccharomyces pombe (2008)
  • Author(s): Yukiko Yoshinari
  • Organizer: 第5回「水と生体分子」公開ワークショップ
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Role of the IXI/V motif in the C-terinal region of StHsp14.0,a small heat shock protein from acidothermophilic archaeon,Sulfolobus tokodaii stain 7,in chaperone function and oligomer assembly (2008)
  • Author(s): Tetsuya Abe
  • Organizer: 第5回「水と生体分子」公開ワークショップ
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Studies on molecular mechanism of group II chaperonin from a hyperthermophilic archaeum,Thermococcus sp.KS-1 (2008)
  • Author(s): Masafumi Yohda
  • Organizer: 第5回「水と生体分子」公開ワークショップ
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] プレフォルディンβサブユニットの結晶構造 (2007)
  • Author(s): 木田 宗志
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-22
• [Presentation] 古細菌型シャペロニンの1分子構造変化計測 (2007)
  • Author(s): 飯塚 怜
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-22
• [Presentation] GroELのC末端欠損変異はATP加水分解反応を遅くする (2007)
  • Author(s): 鈴木 深保子
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] ADPが対称型1:2GroEL-GroES複合体の形成を阻害する (2007)
  • Author(s): 鮫島 知哉
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] 蛍光プローブを用いたGroupII型シャペロニンのhelical protrusionの動的解析 (2007)
  • Author(s): 増田 莉恵
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] グループII型シャペロニンのサブユニット間協同性におけるATPaseの役割 (2007)
  • Author(s): 神前 太郎
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-21
• [Presentation] 蛍光プローブを用いたGroupII型シャペロニンのhelical protrusionの動的解析 (2007)
  • Author(s): 増田 莉恵
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] グループII型シャペロニンのサブユニット間協同性におけるATPaseの役割 (2007)
  • Author(s): 神前 太郎
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] 超好熱性古細菌Thermococcus sp.strain KS-1由来プレフォルディンの機能及び構造解析 (2007)
  • Author(s): 菅野 由利
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] Study the role of interaction between group II chaperonin and prefoldin (2007)
  • Author(s): Muhamad Sahlan
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] 古細菌型シャペロニンの1分子構造変化計測 (2007)
  • Author(s): 飯塚 怜
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] ADPがGroELのtransリングへのATPの結合を阻害することにより2個目のGroESが結合できなくなる (2007)
  • Author(s): 鮫島 知哉
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] GroELのC末端欠損変異はATP加水分解反応を遅くする (2007)
  • Author(s): 鈴木 深保子
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] 分裂酵母Schizosaccharomyces pombe由来sHspの豪性タンパク質との相互作用 (2007)
  • Author(s): 吉成 由貴子
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] Group III鉄含有アルコールデヒドロゲナーゼの大量発現系の構築と酵素活性の検討 (2007)
  • Author(s): 松村 洋寿
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会年会合同大会BMB2007
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] STRUCTURE,FUNCTION AND APPLICATION OF PREFOLDINS FROM HYPERTHERMOPHILIC ARCHAEA (2007)
  • Author(s): Masafumi Yohda
  • Organizer: The 2nd International Workshop on Approaches to Single-Cell Analysis
  • Place of Presentation: Tokyo,Japan
  • Year and Date: 2007-09-07
• [Presentation] The interaction of helical protrusions is important in the protein folding cycle of group II chaperonins (2007)
  • Author(s): Taro Kanzaki
  • Organizer: 3nd Cell Stress Society Intermational Congress on Stress Responses in Biology and Medicine and 2nd World Conference of Stress
  • Place of Presentation: Budapest,Hungary
  • Year and Date: 2007-08-25
• [Presentation] Interaction of prefoldin with group II chaperonin in the presence of various nucleotides (2007)
  • Author(s): Tamotsu Zako
  • Organizer: 3nd Cell Society International Congress on Stress Responses in Biology and Medicine and 2nd World Conference of Stress
  • Place of Presentation: Budapest,Hungary
  • Year and Date: 2007-08-25
• [Presentation] Role of the IXI/V motif in oligomer assembly and function of StHsp14.0,a small heat shock protein from Sulfolobus tokodaii (2007)
  • Author(s): Masafumi Yohda
  • Organizer: 21st Symposium of Protein Society Proteins:From Birth to Death
  • Place of Presentation: Boston,MA
  • Year and Date: 2007-06-22
• [Presentation] グループII型シャペロニンのサブユニット間協調性におけるHelical protrusionの機能 (2007)
  • Author(s): 神前 太郎
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] Construction and characterization of the α_8β_8 hetero-oligomer of group II chaperonin from Thermococcus sp. strain KS-1 (2007)
  • Author(s): Muhamad Sahlan
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] プレフォルディン-シャペロニンの相互作用解析による基質受け渡し機構の解明 (2007)
  • Author(s): 座古 保
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] 超好熱性古細菌Thermococcus sp.strain KS-1由来プレフォルディンの機能解析 (2007)
  • Author(s): 菅野 由利
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] 好酸性好熱古細菌由来StHsp14.0の多量体形成へのC末端IXIモチーフの役割と機能解析 (2007)
  • Author(s): 阿部 哲也
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] 分裂酵母Schizosaccharomyces pombe由来sHspの変性タンパク質との相互作用 (2007)
  • Author(s): 吉成 由貴子
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] マウスPERK小胞体ドメインの大量発現系の構築と精製 (2007)
  • Author(s): 中込 篤
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2007-05-25
• [Presentation] Studies on molecular mechanism for protein folding by group II chaperonins using the chaperonin complexes composed of the wild type and mutant subunits (2007)
  • Author(s): Masafumi Yohda
  • Organizer: VII European Symposium of the Protein Society
  • Place of Presentation: Stockholm,Sweden
  • Year and Date: 2007-05-13
• [Presentation] Two small heat shock proteins of a fission yeast functionin different manner to cope with wide range of temperatures and various denatured proteins (2007)
  • Author(s): Masafumi Yohda
  • Organizer: 3nd Cell Society International Congress on Stress Responses in Biology and Medicine and 2nd World Conference of Stress
  • Place of Presentation: Budapest,Hungary