機能性タンパク質の動力学と折り畳み

• Project/Area Number: 15076208
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Nara Institute of Science and Technology
• Principal Investigator: 片岡 幹雄 Nara Institute of Science and Technology, 物質創成科学研究科, 教授 (30150254)
• Co-Investigator(Kenkyū-buntansha): 山崎 洋一 奈良先端科学技術大学院大学, 物質創成科学研究科, 助教 (40332770)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥75,000,000 (Direct Cost: ¥75,000,000); Fiscal Year 2007: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2006: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2005: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2004: ¥22,000,000 (Direct Cost: ¥22,000,000); Fiscal Year 2003: ¥29,000,000 (Direct Cost: ¥29,000,000)
• Keywords: 生物物理 / 蛋白質 / 動力学 / 折り畳み / 中性子非弾性散乱 / 高角溶液散乱 / 天然変性タンパク質 / 光反応中間体 / CH / π相互作用 / タンパク質動力学 / タンパク質折り畳み / タンパク質の機能 / イェロープロテイン / 黄色ブドウ球菌核酸分解酵素 / ガラス転移 / 中性子非干渉性散乱 / 動力学転移 / バクテリオロドプシン / 時間分解X線回折 / 動的不均一性 / 平均自乗変位 / アレスチン / 会合体形成 / X線溶液散乱 / 光反応

Research Abstract

機能性タンパク質の典型として水溶性光受容タンパク質イェロープロテイン(PYP)および核酸分解酵素(SNase)を用いて、折り畳みおよび機能発現に伴う動力学に関する研究を行った。世界的に重要視されているタンパク質動力学に関する中性子非弾性散乱を用いた研究は、日本では我々のグループだけが行っている。
1.PYP光反応中間体の溶液構造:PYPの活性光反応中間体であるM中間体の溶液構造を高角X線溶液散乱と揺らぎ解析の手法により明らかにした。発色団での光吸収の情報は、α4ヘリックス、β4とβ5のループおよびN端領域に伝播される。NMR溶液構造解析の結果は、必ずしも正確ではなく、局所的な変化を精度よく検出するが、大域的な構造変化に対してはあいまいさが残ることを示した。
2.誘導折り畳みの機構:近年、天然変性タンパク質(IDP)の存在が脚光を浴びている。IDPは、生理的条件下では構造を取らず、標的との結合により折り畳まれる(誘導折り畳み)。我々は誘導折り畳みを示すSNase変異体を多種類創出した。阻害剤による誘導折りたたみの速度論的測定から、D⇔DI→FLの反応に従うものと、D⇔F→FLの反応に従うものが存在することを示した(D、F、Lは変性状態、天然状態、リガンドを表す)。前者は結合がフォールディングに先立って起きると言うモデルである。両方のメカニズムが起きうることを明確に示したのは世界で初めてである。
3.動力学に対する水和効果:中性子非弾性散乱により動力学に対する水和の効果を詳細に調べた。水和の効果は、5meV以下の低エネルギー領域にのみ現れる。低温ではボソンピークと呼ばれる励起モードが現れるが、水和により、ボソンピークの位置は高エネルギー側にシフトした。この結果は、水和水による水素結合がタンパク質を硬くすることを示しており、城地らによる理論からの予測を裏付けるものである。

Research Products

• [Journal Article] Confonnational changes in the N-terminal region of photoactive yellow protein: A time-resolved diffusion study (2008)
  • Author(s): Yuji Hoshihara
  • Journal Title: Biophysical Journal 94 Pages: 2187-2193
  • Peer Reviewed
• [Journal Article] Hydration-dependent protein dynamics revealed by molecular dynamics simulation of crystalline Staphylococcal nuclease. (2008)
  • Author(s): Yasumasa Joti
  • Journal Title: Journal of Physical Chemistry B 112 Pages: 3522-3528
  • Peer Reviewed
• [Journal Article] Diverse roles of glycine residues conserved in photoactive yellow proteins. (2008)
  • Author(s): Yasushi Imamoto
  • Journal Title: Biophysical Journal 94(印刷中)
  • Peer Reviewed
• [Journal Article] Role of the N-tenninal region in the function of the photosynthetic bacterium transcription regulator PpsR. (2008)
  • Author(s): Yoichi Yamazaki
  • Journal Title: Photochemistry and Photobiology (印刷中)
  • Peer Reviewed
• [Journal Article] Mechanism of induced folding: Both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants. (2008)
  • Author(s): Masayoshi Onitsuka
  • Journal Title: Proteins: Structure, Function and Bioinformatics (印刷中)
  • Peer Reviewed
• [Journal Article] Hydration effects on low-frequency protein dynamics observed in simulated neutron scattering spectra. (2008)
  • Author(s): Yasumasa Joti
  • Journal Title: Biophysical Journal 94(印刷中)
  • Peer Reviewed
• [Journal Article] The photoreaction of the photoactive yellow protein domain in the Hght sensor histidine kinase Ppr is influenced by the C-terminal domains. (2008)
  • Author(s): Hironari Kamikubo
  • Journal Title: Photochemistry and Photobiology (印刷中)
  • Peer Reviewed
• [Journal Article] Lowtemperature spectroscopy of Met1OOAla mutant of photoactive yellow protein. (2008)
  • Author(s): Yasushi Imamoto
  • Journal Title: Photochemistry and Photobiology (印刷中)
  • Peer Reviewed
• [Journal Article] Attempt to simplify the aniino-acid sequence of photoactive yellow protein with a set of simple rules. (2007)
  • Author(s): Kumiko Shirai
  • Journal Title: Proteins: Structure, Function and Bioinformatics 67 Pages: 821-833
  • Peer Reviewed
• [Journal Article] Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering. (2007)
  • Author(s): Hironari Kamikubo
  • Journal Title: Biophysical Journal 92 Pages: 3633-3642
  • Peer Reviewed
• [Journal Article] Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy. (2007)
  • Author(s): Koichi Matsuo
  • Journal Title: Biophysical Journal 92 Pages: 4088-4096
  • Peer Reviewed
• [Journal Article] Non-Gaussian behavior of elastic incoherent neutron scattering profiles of proteins studied by molecular dynamics simulation. (2007)
  • Author(s): Atsushi Tokuhisa
  • Journal Title: Physical Review E 75 Pages: 419121-7
  • Peer Reviewed
• [Journal Article] Characterization and crystallization of an IscU-type scaffold protein with bound [2Fe 2S] cluster from the hypertliermophile, Aquifex aeolicus. (2007)
  • Author(s): Yoshimitsu Shimomura
  • Journal Title: Journal of Biochemistry 142 Pages: 577-586
  • Peer Reviewed
• [Journal Article] Attempt to Simplify the Amino-Acid Sequence of Photoactive Yellow Protein with a Set of Simple Rules (2007)
  • Author(s): K.Shirai
  • Journal Title: Proteins : Structu. Funct. Bioinformat. (in press)
• [Journal Article] Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering (2007)
  • Author(s): H.Kamikubo
  • Journal Title: Biophys. J. (in press)
• [Journal Article] Secondary-Structure Analysis of Denatured Proteins by Vacuum-Ultraviolet Circular Dichroism Spectroscopy (2007)
  • Author(s): K.Matsuo
  • Journal Title: Biophys. J. (in press)
• [Journal Article] Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily (2007)
  • Author(s): Y.Imamoto
  • Journal Title: Photochem. Photobiol. (in press)
• [Journal Article] Array of aromatic amino acid side chains located near the chromophore of photoactive yellow protein (2007)
  • Author(s): T.Morishita
  • Journal Title: Photochem. Photobiol. (in press)
• [Journal Article] Preparation of large crystal of photoactive yellow protein for neutron and high resolution crystal structure analysis (2007)
  • Author(s): S.Yamaguchi
  • Journal Title: Photochem. Photobiol. (in press)
• [Journal Article] Time-resolved thermodynamics : Heat capacity change of transient species during photo-reaction of PYP (2006)
  • Author(s): J.S.Kahn
  • Journal Title: J. Am. Chem. Soc. 128 Pages: 1002-1008
• [Journal Article] Stilbene analogs in Hula-twist photoisomerization (2006)
  • Author(s): L.-Y.Yang
  • Journal Title: Photochem. Photobiol. Sci. 5 Pages: 874-882
• [Journal Article] A single CH/π weak hydrogen bond governs stability and photocycle of photoactive yellow protein (2006)
  • Author(s): Y.Imamoto
  • Journal Title: J. Am. Chem. Soc. 128 Pages: 10646-10647
• [Journal Article] Conformational preference of polyglycine in solution to elongated structure (2006)
  • Author(s): S.Ohnishi
  • Journal Title: J. Am. Chem. Soc. 128 Pages: 16338-16344
• [Journal Article] Dynamical heterogeneity of protein dynamics studied by elastic incoherent neutron scattering and molecular simulations (2006)
  • Author(s): H.Nakagawa
  • Journal Title: Materials Science and Engineering A 442 Pages: 356-360
• [Journal Article] Hydration-coupled protein boson peak measured by incoherent neutron scattering (2006)
  • Author(s): H.Nakagawa
  • Journal Title: Physica B 385-386 Pages: 871-873
• [Journal Article] pH-Dependent equilibrium between long-lived near-UV intermediates of photoactive yellow protein (2006)
  • Author(s): N.Shimizu
  • Journal Title: Journal of Biological Chemistry 281(7) Pages: 4318-4325
• [Journal Article] Dynamical heterogeneity of protein dynamics studied by elastic incoherent neutron scattering and molecular simulations (2006)
  • Author(s): H.Nakagawa
  • Journal Title: Materials Science and Engineering (印刷中)
• [Journal Article] Cloning and characterization of nif structural and regulatory genes in the purple sulfur bacterium, Halorhodospira halophila (2006)
  • Author(s): H.Tsuihiji
  • Journal Title: J. Biosci. Bioeng (印刷中)
• [Journal Article] The Crystal Structure of the R52Q Mutant Demonstrates a Role for R52 in Chromophore pK_a Regulation in Photoactive Yellow Protein (2006)
  • Author(s): N.Shimizu
  • Journal Title: Biochemistry (印刷中)
• [Journal Article] A biosensor in the time domain based on the diffusion coefficient measurement : intermolecular interaction of an intermediate of photoactive yellow protein. (2005)
  • Author(s): J.S.Khan
  • Journal Title: Anal. Chem. 77 Pages: 6625-6629
• [Journal Article] タンパク質と水の中性子散乱 (2005)
  • Author(s): 山口敏男
  • Journal Title: 現代化学 411 Pages: 55-60
• [Journal Article] Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base : Microsecond time-resolved X-ray diffraction study of purple membrane (2005)
  • Author(s): Toshihiko Oka
  • Journal Title: Biophysical Journal 88 Pages: 436-442
• [Journal Article] Protein Dynamical Heterogeneity Derived from Neutron Incoherent Elastic Scattering (2004)
  • Author(s): Hiroshi Nakagawa
  • Journal Title: Journal of Physical Society of Japan 73 Pages: 491-495
• [Journal Article] Time and frequency domain investigations on ultrafast photoreaction dynamics of photoactive yellow protein (2004)
  • Author(s): Haik Chosrowjan
  • Journal Title: Review of Laser Engineering 32 Pages: 114-120
• [Journal Article] Raman Spectroscopy Reveals the Origin of an Intermediate Wavelength Form in Photoactive Yellow Protein (2004)
  • Author(s): S.F.El-Mashtoly
  • Journal Title: Biochemistry 43 Pages: 2279-2287
• [Journal Article] Picometer-scale dynamical observations of individual membrane proteins : The case of bacteriorhodopsin (2004)
  • Author(s): Y.Okumura
  • Journal Title: Physical Review E 70 Pages: 21917-21917
• [Journal Article] Direct observation of the pH-dependent equilibrium between L-like and M intermediates of photoactive yellow protein (2004)
  • Author(s): Yasushi Imamoto
  • Journal Title: FEBS Letters 577 Pages: 75-80
• [Presentation] 中性子散乱によるタンパク質動力学の研究 (2008)
  • Author(s): 片岡幹雄
  • Organizer: 第55回応用物理学関係連合講演会
  • Place of Presentation: 船橋
  • Year and Date: 2008-03-27
• [Presentation] Structure of the photointermediate of PYP and the propagation mechanism of structural change. (2008)
  • Author(s): 片岡幹雄
  • Organizer: Gordon Research Conference on Photosensory Receptors & Signal Transduction
  • Place of Presentation: ヴェンチュラ(アメリカ合衆国カリフォルニア州)
  • Year and Date: 2008-01-30
• [Presentation] ジヒドロ葉酸還元酵素におけるフォールディングエレメントや活性部位の接続部位に含まれる情報の抽出 (2007)
  • Author(s): 政さやか
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-23
• [Presentation] 網羅的アラニン挿入変異法によるジヒドロ葉酸還元酵素の構造形成および機能発現領域の抽出 (2007)
  • Author(s): 芝るみ
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-23
• [Presentation] Ala/ValスキヤニングによるPhotoactive Yellow Proteinの構造・機能に対する重要なアミノ酸残基のマツピング (2007)
  • Author(s): 永田大介
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-23
• [Presentation] Photoactive Yellow Proteinを用いた中性子結晶構造解析 (2007)
  • Author(s): 山口繁生
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-23
• [Presentation] イェロープロテインの光反応サイクルにおける構造変化メカニズム (2007)
  • Author(s): 今元泰
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-23
• [Presentation] C末端欠損スタフイロコツカルヌクレアーゼが示す誘導折り畳み反応の分子メカニズム (2007)
  • Author(s): 鬼塚正義
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-22
• [Presentation] アラニン挿入変異が折れ畳み過程に及ぼす影響 (2007)
  • Author(s): 澤田宏起
  • Organizer: 第45回生物物理学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-22
• [Presentation] Effect of hydration on the glass-like transition of protein. (2007)
  • Author(s): 片岡幹雄
  • Organizer: Fukuoka International Workshop on Unifying Concepts of Glass Transition
  • Place of Presentation: 福岡
  • Year and Date: 2007-11-23
• [Presentation] Solution structural analysis of GGA(VHS-GAT) and the complexes with its binding partners using solution X-ray scattering. (2007)
  • Author(s): 上久保裕生
  • Organizer: The 9th International Conference on Biology and Synchrotron Radiation
  • Place of Presentation: マンチェスター(連合王国)
  • Year and Date: 2007-08-15
• [Presentation] Wide angle X-ray solution scattering reveals the structure of photointennediate of photoactive yellow protein. (2007)
  • Author(s): 片岡幹雄
  • Organizer: The 9th International Conference on Biology and Synchrotron Radiation
  • Place of Presentation: マンチェスター(連合王国)
  • Year and Date: 2007-08-15
• [Presentation] Determination of hydrogen positions of photoactive yellow protein. (2007)
  • Author(s): 山口繁生
  • Organizer: The 9th International Conference on Biology and Synchrotron Radiation
  • Place of Presentation: マンチェスター(連合王国)
  • Year and Date: 2007-08-15
• [Presentation] Structure of the photointermediate of PYP revealed by wide-angle x-ray solution scattering and fluctuation analysis. (2007)
  • Author(s): 片岡幹雄
  • Organizer: Telluride Conference on Protein Dynamics
  • Place of Presentation: テルライド(アメリカ合衆国コロラド州)
  • Year and Date: 2007-07-31
• [Book] タンパク質科学-構造・物性・機能 (2005)
  • Author(s): 片岡幹雄(分担執筆)
  • Total Pages: 579
  • Publisher: 化学同人
• [Remarks]
  • URL: http://mswebs.naist.jp/LABs/kataoka/index.html
• [Publications] N.Mataga: "Ultrafast photoreactions in protein nanospaces as revealed by fs fluorescence dynamics measurements on photoactive yellow protein and related systems."Phys.Chem.Chem.Phys.. 5. 2454-2460 (2003)
• [Publications] Y.Imamoto: "Concentration-dependent tetramerization of bovine visual arrestin."Biophys.J.. 85. 1186-1195 (2003)
• [Publications] Y.Imamoto: "Photoisomerization by hula twist : 2, 2'-dimethylstilbene and a ring-fused analogue."Angew.Chem.Int.Ed.. 42. 3630-3633 (2003)
• [Publications] M.Arai: "Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase."J.Mol.Biol.. 329. 779-791 (2003)
• [Publications] N.Harigai: "Role of an N-terminal loop in the secondary structural change of photoactive yellow protein."Biochemistry. 42. 13893-13900 (2003)
• [Publications] H.Nakagawa: "Protein Dynamical Heterogeneity Derived from Neutron Incoherent Elastic Scattering."J.Phys.Soc.Jpn.. 73(in press). (2004)
• [Publications] 中川 洋: "タンパク質動力学研究における中性子とX線の役割"波紋. 13. 116-119 (2003)