Elucidation of the denatured structure of protein in equilibrium with the native one under a physiological condition.

• Project/Area Number: 21570173
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Kwansei Gakuin University
• Principal Investigator: SEGAWA Shin-ichi 関西学院大学, 理工学部, 教授 (70103132)
• Co-Investigator(Kenkyū-buntansha): NODA Yasuo 関西学院大学, 理工学部, 教育技術主事 (40268511) ; YUTANI Katsuhide 独立行政法人理化学研究所, 理化学研究所播磨研究所, 上級研究員 (90089889)
• Co-Investigator(Renkei-kenkyūsha): SUGIMOTO Hayuki 関西学院大学, 理工学研究科, 博士研究員 (60529527)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2011: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2009: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: タンパク質のフォールディング / H/D交換反応とNMR測定 / リゾチームS-S結合欠損変異体 / 超好熱菌由来のタンパク質 / ピロリドンカルボキシルペプチダーゼ / タンパク質の変性状態 / グリセロールによる選択的水和 / タンパク質フォールディング / 超好熱菌由来タンパク質 / H / D交換反応とNMR測定

Research Abstract

We have studied partially ordered structures remaining in unstructured disulfide-deficient variants of lysozyme by means of NMR and H/D exchange methods. In this residual structure, two residues of I55 and L56 involved in the bottom of the α-sheet were located in a hydrophobic pocket formed with A, B and C-helices in the β-domain. Lysozyme folding appears to be initiated around this site. On the other hand, H/D exchange reactions were studied in the native state of pyrrolidone carboxyl peptidase(PCP). The native structure was found to be divided into two domains by structural changes near at the residue 70.

Research Products

• [Journal Article] Glycerol-Enhanced Detection of a Preferential Structure Latent in Unstructured 1SS-Variants of Lysozyme (2012)
  • Author(s): Noda, Y., Narama, K., Kasai, K., Tachibana, H. & Segawa, S.
  • Journal Title: Biopolymers Volume: 97 Pages: 539-549
  • Peer Reviewed
• [Journal Article] Modulation of Structure and Dynamics by Disulfide Bond Formation in Unfolded States (2012)
  • Author(s): Silvers, R., Sziegat, F., Tachibana, H., Segawa, S., Whittaker, S., Gunther, U. L., Gabel, F., Huang, J., Blackledge, M., Wirmer-Bartoschek, J., Schwalbe, H.
  • Journal Title: J. Am. Chem. Soc Volume: 134 Pages: 6846-6854
  • Peer Reviewed
• [Journal Article] NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase (2012)
  • Author(s): Miyamoto, Y., Noda, Y., Iida, T., Yamaguchi, K., Nishimura, S., Tanaka, A., Segawa, S. & Inui, T.
  • Journal Title: J. Biochem Volume: 151 Pages: 335-342
  • NAID: 10031166072
  • Peer Reviewed
• [Journal Article] NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase (2012)
  • Author(s): Miyamoto, Y., Noda, Y., Iida, T., Yamaguchi, K., Nishimura, S., Tanaka, A., Segawa, S., Inui, T.
  • Journal Title: The Journal of Biochemistry Volume: 151 Issue: 3 Pages: 335-342
  • DOI: 10.1093/jb/mvr140
  • Peer Reviewed
• [Journal Article] Modulation of Structure and Dynamics by Disulfide Bond Formation in Unfolded States (2012)
  • Author(s): Segawa, S., Silvers, R.
  • Journal Title: J.Am.Chem.Soc. Volume: 134 Issue: 15 Pages: 6846-6854
  • DOI: 10.1021/ja3009506
  • Peer Reviewed
• [Journal Article] NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase (2011)
  • Author(s): Sugimoto, H., Noda, Y. & Segawa, S.
  • Journal Title: J. Mol. Biol Volume: 412 Pages: 304-315
  • Peer Reviewed
• [Journal Article] NMR analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase (2011)
  • Author(s): Segawa, S., Sugimoto, H.
  • Journal Title: J.Mol.Biol. Volume: 412 Issue: 2 Pages: 304-315
  • DOI: 10.1016/j.jmb.2011.07.025
  • Peer Reviewed
• [Journal Article] Glycerol-Induced Folding of Unstructured Disulfide-Deficient Lysozyme into a Native-Like Conformation (2009)
  • Author(s): Sakamoto, K., Hirai, K., Kitamura, Y., Yamazaki, K., Yusa, M., Tokunaga, N., Doi, G., Noda, Y., Tachibana, H. & Segawa, S.
  • Journal Title: Biopolymers Volume: 91 Pages: 665-675
  • Peer Reviewed
• [Journal Article] Glycerol-Induced Folding of Unstructured Disulfide-Deficient Lysozyme into a Native-Like Conformation (2009)
  • Author(s): Sakamoto, K.
  • Journal Title: Biopolymers 91 Pages: 665-675
  • Peer Reviewed
• [Presentation] ピロリドンカルボキシルペプチダーゼの折りたたみ-天然類似の反応中間体の検出 (2012)
  • Author(s): キンマン・クリストファー、飯村哲史、野田康夫、油谷克英、瀬川新一
  • Organizer: 日本物理学会第67回年次大会
  • Place of Presentation: 関西学院大学、西宮
  • Year and Date: 2012-03-25
• [Presentation] ピロリドンカルボキシルペプチダーゼの天然立体構造の大規模な揺らぎをH/D交換反応で検出する (2012)
  • Author(s): 薮本和義、飯村哲史、野田康夫、油谷克英、瀬川新一
  • Organizer: 日本物理学会第67回年次大会
  • Place of Presentation: 関西学院大学、西宮
  • Year and Date: 2012-03-25
• [Presentation] ピロリドンカルボキシルペプチダーゼの折りたたみ-天然類似の反応中間体の検出 (2012)
  • Author(s): キンマン・クリストファー
  • Organizer: 日本物理学会第67回年次大会
  • Place of Presentation: 関西学院大学西宮上ケ原キャンパス、兵庫県
  • Year and Date: 2012-03-25
• [Presentation] ピロリドンカルボキシルペプチダーゼの天然立体構造の大規模な揺らぎをH/D交換反応で検出する (2012)
  • Author(s): 薮本和義
  • Organizer: 日本物理学会第67回年次大会
  • Place of Presentation: 関西学院大学西宮上ケ原キャンパス、兵庫県
  • Year and Date: 2012-03-25
• [Presentation] S-S結合欠損リゾチームを用いた折りたたみ反応研究の総括-折りたたみ反応における各S-S結合の役割 (2011)
  • Author(s): 瀬川新一、奈良間邦成、野田康夫、橘秀樹
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: ホテル阪急エキスポパーク、大阪
  • Year and Date: 2011-06-09
• [Presentation] S-S結合欠損リゾチームを用いた折りたたみ反応研究の総括-折りたたみ反応における各S-S結合の役割 (2011)
  • Author(s): 瀬川新一
  • Organizer: 第11回日本蛋白質科学会
  • Place of Presentation: ホテル阪急エキスポパーク、大阪府
  • Year and Date: 2011-06-09
• [Presentation] ピロリドンカルボキシルペプチダーゼ(PCP)の天然構造の揺らぎと折りたたみ反応過程の構造との関連 (2011)
  • Author(s): 薮本和義
  • Organizer: 第11回日本蛋白質科学会
  • Place of Presentation: ホテル阪急エキスポパーク、大阪府
  • Year and Date: 2011-06-08
• [Presentation] Glycerol-induced folding in four species of 1SS-variants of lysozyme-the potential folded structure remaining in unstructured proteins. (2010)
  • Author(s): Narama, K.
  • Organizer: 第48回日本生物物理学会
  • Place of Presentation: 東北大学、川内キャンパス
  • Year and Date: 2010-09-21
• [Presentation] Structural stability of each domain appearing in the early stage of PCP folding monitored by amide H/D exchange rates. (2010)
  • Author(s): Yabumoto, K.
  • Organizer: 第48回日本生物物理学会
  • Place of Presentation: 東北大学、川内キャンパス
  • Year and Date: 2010-09-20
• [Presentation] NMR Analysis of a Kinetically Trapped Refolding Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase (2010)
  • Author(s): Sugimoto, H., Noda, Y. & Segawa, S.
  • Organizer: 24^<th> Annual Symposium of The Protein Society
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2010-08-04
• [Presentation] NMR Analysis of a Kinetically Trapped Refolding Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase. (2010)
  • Author(s): Sugimoto, H.
  • Organizer: 24^<th> Annual Symposium of The Protein Society
  • Place of Presentation: San Diego, CA, USA.
  • Year and Date: 2010-08-04
• [Presentation] ジスルフィド結合を欠損させたデンプン結合ドメインのリフォールディング中間体のNMR解析 (2010)
  • Author(s): 杉本華幸、野田康夫、瀬川新一
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2010-06-17
• [Presentation] H/D交換NMR測定法によるピロリドンカルボキシルペプチダーゼ(PCP)の折りたたみ反応における逐次構造変化の研究 (2010)
  • Author(s): キンマンクリストファー、薮本和義、横田洋平、野田康夫、油谷克英、瀬川新一
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2010-06-16
• [Presentation] NMR分光法を用いたジスルフィド結合を欠損させたデンプン結合ドメインのリフォールディング中間体の構造解析 (2010)
  • Author(s): 杉本華幸
  • Organizer: 日本農芸化学会2010年度大会
  • Place of Presentation: 東京大学駒場キャンパス
  • Year and Date: 2010-03-29
• [Presentation] Residual Nonrandom Structures in Disulfide-Deficient Variants of Lysozyme (2009)
  • Author(s): Kasai, K.
  • Organizer: 第47回日本生物物理学会
  • Place of Presentation: アスティとくしま
  • Year and Date: 2009-10-30
• [Presentation] Sequential folding pathway of PCP(pyrrolidone carboxyl peptidase)in the molten-globule-like stage and its refolding to the native state. (2009)
  • Author(s): Yokota, Y.
  • Organizer: 第47回日本生物物理学会
  • Place of Presentation: アスティとくしま
  • Year and Date: 2009-10-30
• [Presentation] NMR analysis of a kinetically trapped refolding intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase (2009)
  • Author(s): 杉本華幸、野田康夫、瀬川新一
  • Organizer: 第82回日本生化学会
  • Place of Presentation: 神戸ポートアイランド
  • Year and Date: 2009-10-22
• [Presentation] NMR analysis of intermediate in thermal unfolding process of mouse lipocalin-type prostaglandin D synthase (2009)
  • Author(s): Miyamoto, Y.
  • Organizer: 第82回日本生化学会
  • Place of Presentation: 神戸ポートアイランド
  • Year and Date: 2009-10-22
• [Presentation] The folding of PCP(Pyrrolidone Carboxyl Peptidase) proceeds in the denatured state similar to the stage of molten globule (2009)
  • Author(s): Segawa, S., Yokota, Y., Dosei, M., Noda, Y. & Yutani, K.
  • Organizer: The 23^<rd> Symposium of The Protein Society
  • Place of Presentation: Boston, U. S. A.
  • Year and Date: 2009-07-25
• [Presentation] The folding of PCP(Pyrrolidone Carboxyl Peptidase)proceeds in the denatured state similar to the stage of molten globule (2009)
  • Author(s): Segawa, S.
  • Organizer: The 23rd Symposium of The Protein Society
  • Place of Presentation: Boston, MA, U.S.A.
  • Year and Date: 2009-07-25
• [Remarks]