蛋白質中に存在する低障壁水素結合の特性解析

• Project Area: Molecular Science for Supra Functional Systems ? Development of Advanced Methods for Exploring Elementary Process
• Project/Area Number: 22018019
• 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: 上久保 裕生 奈良先端科学技術大学院大学, 物質創成科学研究科, 准教授 (20311128)
• Project Period (FY): 2010 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,000,000 (Direct Cost: ¥4,000,000); Fiscal Year 2011: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 2010: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: 中性子結晶構造解析 / 水素結合 / 低障壁水素結合 / 光センサー蛋白質

Research Abstract

本研究では、低障壁水素結合(LBHB)の存在が実証された光センサー蛋白質(Photoactive Yellow Protein, PYP)をモデル蛋白質として、蛋白質中でのLBHBの物理化学的特性を明らかにすることを目的とし実施してきた。本年度は、(1)LBHBの結合エネルギーの評価を行い、(2)LBHBの形成条件について検証した。
(1)LBHBを通常の水素結合に変換した変異体(E46Q)を作製し、X線並びに中性子併用結晶構造解析によって変異効果を検証した。変異に伴い、LBHBに近接する短距離水素結合(pCA-Y42)の水素結合長が、2.52Aから2.48Aまで短くなっているものの、pCA-Y42間の水素原子はY42と共有結合しており、イオン性水素結合が保持されていることが確認できた。これらの水素結合の変化を除き、他の領域では、顕著な構造変化を示さなかったことを踏まえ、野生型 (LBHB)、E46Q (HB)及びE46A (水素結合なし)に対して酸滴定を行い、LBHBの結合自由エネルギー(6.9 kcal/mol)は、HB (4.4kcal/mol)の約1.5倍程度であることを明らかにした。(2)(1)の結果から、短距離イオン性水素結合(pCAごY42)が、低障壁水素結合形成に作用していることが示唆された。検証のためpCA-Y42間の水素結合を解消した変異体(Y42F)の構造解析を行ったところ、置換に伴い、pCA-Y/F42のC_ξ原子間距離が3.24Aから3.29Aに増加すると同時に、pCA-E46間の水素結合長は2.56Aから2.63Aまで増加することがわかった。この結果は、置換に伴い、pCA-E46間の低障壁水素結合が通常の水素結合に転換したことを示している。以上の結果から、pCA-E46間の低障壁水素結合が形成に際する、pCA-Y42間のイオン性水素結合による幾何学的制限の重要性を示している。

Research Products

• [Journal Article] Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect ^<13>C NMR spectroscopy (2011)
  • Author(s): N.A.Oktaviani, T.J.Pool, H.Kamikubo, J.Slager, R.M.Sceek, M.Kataoka, F.A.A.Mulder
  • Journal Title: Biophys.J. Volume: 102 Pages: 579-586
  • Peer Reviewed
• [Journal Article] Changes in the hydrogen-bond network around the chromophore of photoactive yellow protein in the ground and excited states (2011)
  • Author(s): M.Mizuno, H.Kamikubo, M.Kataoka, Y.Mizutani
  • Journal Title: J.Phys.Chem.B Volume: 115 Pages: 9306-9310
  • Peer Reviewed
• [Journal Article] Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase (2011)
  • Author(s): Rumi Shiba
  • Journal Title: BIOPHYSICS Volume: 7 Pages: 1-10
  • Peer Reviewed
• [Journal Article] Effect of conformational states on protein dynamical transition (2010)
  • Author(s): Hiroshi Nakagawa
  • Journal Title: Biochim.Biophys.Acta Volume: 1804 Pages: 27-33
  • Peer Reviewed
• [Journal Article] Non-local interaction responsible for the tertiary structural formation of Staphylococcal nuclease (2010)
  • Author(s): Shingo Kato
  • Journal Title: Biophys.J. Volume: 98 Pages: 678-786
  • Peer Reviewed
• [Journal Article] Self-Assembly and Enhanced Magnetic Properties of Three-dimensional Superlattice Structures Composed of Cube- and Sphere-Shaped EuS Nanocrsytals (2010)
  • Author(s): Atsushi Tanaka
  • Journal Title: Chem.Mat. Volume: 22 Pages: 1776-1781
  • Peer Reviewed
• [Journal Article] Cytochrome c polymerization by successive domain swapping at the C-terminal helix (2010)
  • Author(s): Shun Hirota
  • Journal Title: Proc.Natl.Acad.Sci.USA Volume: 107 Pages: 12854-12859
  • Peer Reviewed
• [Journal Article] Size-controlled aggregation of cube-shaped EuS nanocrystals with magneto-optic properties in solution (2010)
  • Author(s): Atsushi Tanaka
  • Journal Title: Langmuir Volume: 27 Pages: 104-108
  • Peer Reviewed
• [Journal Article] フォトアクティブイェロープロテインの中性子結晶構造解析 (2010)
  • Author(s): 上久保裕生
  • Journal Title: RADIOISOTOPES Volume: 59 Pages: 289-297
  • NAID: 10026244827
  • Peer Reviewed
• [Presentation] Geometric Restriction Facilitates LBHB Formation in Photoactive Yellow Protein (2012)
  • Author(s): Y.Nishiguchi, H.Kamikubo, S.Yamaguchi, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: The 5th International Symposium on Molecular Science of Fluctuations toward Biological Functions
  • Place of Presentation: 奈良県奈良市
  • Year and Date: 2012-01-08
• [Presentation] The Role of N-terminal Region for Photoreaction of Various Photoactive Yellow Proteins (2012)
  • Author(s): R.Odani, Y.Yamazaki, K.Matsumoto, M.Yamaguchi, H.Kamikubo, M.Kataoka
  • Organizer: The 5th International Symposium on Molecular Science of Fluctuations toward Biological Functions
  • Place of Presentation: 奈良県奈良市
  • Year and Date: 2012-01-08
• [Presentation] Low barrier hydrogen bond in photoactive yellow protein and its role in photoreaction (2012)
  • Author(s): H.Kamikubo, S.Yamaguchi, Y.Nishiguchi, K.Yonezawa, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: The 5th International Symposium on Molecular Science of Fluctuations toward Biological Functions
  • Place of Presentation: 奈良県奈良市
  • Year and Date: 2012-01-08
• [Presentation] 蛋白質中に存在する低障壁水素結合の特性解析 (2011)
  • Author(s): 上久保裕生
  • Organizer: 特定領域研究「分子高次系機能解明のための分子科学」第4回公開シンポジウム
  • Place of Presentation: 宮城県仙台市(招待講演)
  • Year and Date: 2011-11-25
• [Presentation] Physicochemical properties of low barrier hydrogen bonds involved in Photoactive Yellow Protein (2011)
  • Author(s): H.Kamikubo, M.Kataoka
  • Organizer: 1st Asia-Oceania Conference on Neutron Scattering
  • Place of Presentation: 茨城県つくば市(招待講演)
  • Year and Date: 2011-11-24
• [Presentation] The roles of the low barrier hydrogen bond involved in Photoactive Yellow Protein (2011)
  • Author(s): H.Kamikubo, S.Yamaguchi, Y.Nishiguchi, K.Yonezawa, K.Kurihara, T.Tamada, M.Kataoka
  • Organizer: 17th International Biophysics Congress (IUPAB)
  • Place of Presentation: Beijing, China
  • Year and Date: 2011-11-02
• [Presentation] Comparison of structural elements between Staphylococcal nuclease and the C-terminal domain of Human p100 (2011)
  • Author(s): S.Ueyanagi, H.Kamikubo, Y.Ogawa, C.Komeda, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: 17th International Biophysics Congress (IUPAB)
  • Place of Presentation: Beijing, China
  • Year and Date: 2011-11-02
• [Presentation] Local structural change of staphylococcal nuclease wild type and its non-native mutant with guanidine hydrochloride monitored by tryptophan triplet quenching (2011)
  • Author(s): M.Yamaguchi, Y.Yamazaki, H.Kamikubo, M.Kataoka
  • Organizer: 17th International Biophysics Congress (IUPAB)
  • Place of Presentation: Beijing, China
  • Year and Date: 2011-11-02
• [Presentation] Refinement of function element of dihydrofolate reductase by the systematic alanine insertion (2011)
  • Author(s): R.Shiba, H.Kamikubo, M.Umeyama, S.Tsukasa, Y.Yamazaki, M.Yamaguchi, M.Iwakura, M.Kataoka
  • Organizer: 17th International Biophysics Congress (IUPAB)
  • Place of Presentation: Beijing, China
  • Year and Date: 2011-11-02
• [Presentation] Phisicochemical properties of low barrier hydrogen bonds involved inproteins (2011)
  • Author(s): H.Kamikubo
  • Organizer: 2011 GIST-NAIST-NCTU Joint Symposium on Advanced Materials
  • Place of Presentation: Gwangju, Korea(招待講演)
  • Year and Date: 2011-10-31
• [Presentation] 活性部位近傍に存在する水素結合と高次構造転移 (2011)
  • Author(s): 上久保裕生
  • Organizer: 特定領域研究「高次系分子科学」第12回ミニ公開シンポジウム「光受容蛋白質」
  • Place of Presentation: 愛知県蒲郡市(招待講演)
  • Year and Date: 2011-10-27
• [Presentation] Attempt of functional modification of a protein by element implantation (2011)
  • Author(s): E.Sakai, H.Kamikubo, A.Kogasaka, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] Structural Analysis of Intrinsically Disordered Protein Model using FRET (2011)
  • Author(s): T.Muto, Y.Kishi, M.Yamaguchi, K.Shiraga, I.Iijima, Y.Yamazaki, H.Kamikubo, T.Hosaka, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] Development of Light dependent protein activity control system by use of interaction of the Rc-PYP (2011)
  • Author(s): S.Setoguchi, H.Kubo, Y.Yamazaki, H.Kamikubo, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] The Role of N-terminal Region for Photoreaction of Various Photoactive Yellow Proteins (2011)
  • Author(s): R.Odani, Y.Yamazaki, K.Matsumoto, H.Kamikubo, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] Analysis of low barrier hydrogen bond in Photoactive Yellow Protein, Geometric restriction facilitates LBHB formation in PYP (2011)
  • Author(s): Y.Nishiguchi, H.Kamikubo, S.Yamaguchi, T.Tamada, K.Kurihara, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] Solution structure of the complex states of Rhodobacter capsulatus PYP with its binding protein (2011)
  • Author(s): Y.Yamazaki, M.Hayashi, H.Kamikubo, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-18
• [Presentation] Cytochrome c polymerization by domain swapping (2011)
  • Author(s): S.Nagao, Y.Hattori, M.Ueda, M.Taketa, H.Osuka, H.Komori, H.Kamikubo, S.Negi, Y.Sugiura, M.Kataoka, Y.Higuchi, S.Hirota
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-17
• [Presentation] Denatured Structure of Staphylococcal Nuclease Studied by Triplet State Quenching of Tryptophan by Cysteine (2011)
  • Author(s): T.Okada, M.Yamaguchi, Y.Yamazaki, H.Kamikubo, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-17
• [Presentation] Comparison of structural elements between Staphylococcal nuclease and the C-terminal domain of Human p100 (2011)
  • Author(s): S.Ueyanagi, H.Kamikubo, Y.Ogawa, C.Komeda, Y.Yamazaki, M.Yamaguchi, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-16
• [Presentation] Classification of function element of dihydrofolate reductase by the systematic alanine insertion (2011)
  • Author(s): R.Shiba, H.Kamikubo, M.Umeyama, S.Tsukasa, Y.Yamazaki, M.Yamaguchi, Ma.Iwakura, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-16
• [Presentation] Difference in local structure between staphylococcal nuclease wild type and its non-native mutant studied by tryptophan triplet quenching (2011)
  • Author(s): M.Yamaguchi, Y.Yamazaki, H.Kamikubo, M.Kataoka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市
  • Year and Date: 2011-09-16
• [Presentation] 構造生物学における放射光X線溶液散乱 (2011)
  • Author(s): 上久保裕生
  • Organizer: 合同PF研究会「PFにおけるマイクロビームを利用したXAFS,XRP,SAXS実験の展望」
  • Place of Presentation: 茨城県つくば市(招待講演)
  • Year and Date: 2011-09-07
• [Presentation] Physicochemical properties of low barrier hydrogen bonds (2011)
  • Author(s): H.Kamikubo
  • Organizer: 特定領域研究「分子高次系機能解明のための分子科学」第5回国際公開シンポジウム
  • Place of Presentation: 北海道札幌市(招待講演)
  • Year and Date: 2011-07-13
• [Presentation] Fluctuation controls enzymatic activity of Staphylococcal nuclease (2010)
  • Author(s): Hironari Kamikubo, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-12-01
• [Presentation] Classification of the functional element of dihydrofolate reductase by the systematic alanine insertion (2010)
  • Author(s): Rumi Shiba, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-12-01
• [Presentation] FRET analysis of structural changes in staphylococcal nuclease (2010)
  • Author(s): Yusuke Kishi, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-12-01
• [Presentation] Cytochrome c polymerization by successive domain swapping at the C-terminal helix (2010)
  • Author(s): Zhonghua Wang, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-12-01
• [Presentation] The investigation of the relationship between non-local interaction and the effect of single alanine insertion in staphylococcal nuclease (2010)
  • Author(s): Hiroki Sawada, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-11-30
• [Presentation] Measurements of loop formation in the denatured state of staphylococcal nuclease (2010)
  • Author(s): Mariko Yamaguchi, et al.
  • Organizer: The 4th International Symposium "Molecular Science of Fluctuations toward Biological Functions"
  • Place of Presentation: 大津
  • Year and Date: 2010-11-30
• [Presentation] Cytochrome c polymerization by successive domain swapping at the C-terminal helix (2010)
  • Author(s): Shun Hirota, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-22
• [Presentation] A case of EspB from enterohaemorrhagic Escherichia coli (2010)
  • Author(s): Daizo Hamada, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-22
• [Presentation] Property of a structural element of SNase-like domain in human p100 (2010)
  • Author(s): Yoko Ogawa, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-22
• [Presentation] The investigation of the relationship between non-local interaction and the effect of single alanine insertion in staphylococcal nuclease (2010)
  • Author(s): Hiroki Sawada, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-22
• [Presentation] FRET analysis of structural changes in Staphylococcal nuclease (2010)
  • Author(s): Yusuke Kishi, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-22
• [Presentation] Solution structure of the light sensor histidine kinase of Ppr comprised of the PYP and Bph domains (2010)
  • Author(s): Junko Miura, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] Solution structural analysis of Drosophila GGA (2010)
  • Author(s): Maiko Naruse, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] Analysis of solution state of the interaction protein of Rc-PYP (2010)
  • Author(s): Miki Hayashi, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] The role of each residue in the flexible Ω loop of Staphylococcal nuclease on the catalytic activity (2010)
  • Author(s): Changjun Ahn, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] Classification of the functional element of dihydrofolate reductase (DHFR) by the systemic alanine insertion (2010)
  • Author(s): Rumi Shiba, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] Synthesis of an artificial enzyme by implanting the functional elements of SNase (2010)
  • Author(s): Hironari Kamikubo, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-21
• [Presentation] Urea denaturation of staphylococcal nuclease monitored by tryptophan-cysteine distance (2010)
  • Author(s): Mariko Yamaguchi, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-20
• [Presentation] Substitution effects of basic residues in the photoactive yellow protein of Rhodobacter caplsulatus (2010)
  • Author(s): Yoichi Yamazaki, et al.
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2010-09-20
• [Presentation] photoactive Yellow Proteinにおける発色団の機能変調 (2010)
  • Author(s): 山崎洋一, et al.
  • Organizer: 第16回日本光生物学協会年会
  • Place of Presentation: 吹田
  • Year and Date: 2010-08-11
• [Presentation] The effect of alanine insertion mutation for folding process. (2010)
  • Author(s): Hiroki Sawada, et al.
  • Organizer: The 4th Shanghai International Conference on Biophysics and Molecular Biology
  • Place of Presentation: Shanghai-Jiashan, China
  • Year and Date: 2010-08-09
• [Presentation] Extraction of the regions encoded foldability and/or functionability from dihydrofolate reductase by the systematic alanine insertion (2010)
  • Author(s): Rumi Shiba, et al.
  • Organizer: The 4th Shanghai International Conference on Biophysics and Molecular Biology
  • Place of Presentation: Shanghai-Jiashan, China
  • Year and Date: 2010-08-09
• [Presentation] 精子由来蛋白質IZUMO,その構造と受精膜融合での役割 (2010)
  • Author(s): 萩原義久, et al.
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 札幌
  • Year and Date: 2010-06-18
• [Presentation] 網羅的アラニン挿入変異解析法を用いたジヒドロ葉酸還元酵素の機能発現及び構造形成領域の抽出 (2010)
  • Author(s): 芝るみ, et al.
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 札幌
  • Year and Date: 2010-06-16
• [Book] 入門 構造生物学(編集:加藤龍一)(2章3節X線溶液散乱) (2010)
  • Author(s): 上久保裕生
  • Total Pages: 8
  • Publisher: 共立出版