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1999 Fiscal Year Final Research Report Summary

Molecular Mechanism of Functional Expression of the Chaperonin

Research Project

Project/Area Number 10480177
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionThe University of Tokyo

Principal Investigator

KUWAJIMA Kunihiro  University of Tokyo ; Graduate School of Science, Professor, 大学院・理学系研究科, 教授 (70091444)

Co-Investigator(Kenkyū-buntansha) ARAI Munehito  University of Tokyo; Graduate School of Science, Research Associate, 大学院・理学系研究科, 助手 (90302801)
Project Period (FY) 1998 – 1999
KeywordsMolecular chaperone / chaperonin / GroEL / ES / Protein folding / α-lactalbumin / staphylococcal nuclease / green fluorescent protein / ATP
Research Abstract

The objective of the present study is to elucidate the molecular mechanism of functional expression of the Escherichia coli chaperonin GroEL/ES. For this purpose, we investigated the interactions of the chaperonin with folding intermediates of three globular proteins, α-lactalbumin (αLA), staphylococcal nuclease (SNase) and green fluorescent protein (GFP), which have very different physical properties. We also investigated physicochemically the interactions of GroEL with the nucleotides (ATP and ADP) that are indispensable for the chaperonin function and the analogs of the nucleotides (ATPγS and AMP-PNP). The following results were obtained.
(1) We used an SNase mutant, in which the refolding kinetics were significantly simplified, and apo-αLA that showed a simple single relaxation kinetics of refolding, as model target proteins of GroEL. We studied the effect of the chaperonin on the refolding kinetics of these target proteins by stopped-flow fluorescence spectroscopy. Especially for α … More LA, we succeeded the quantitative analysis of the reaction curves by computer simulations. When we added a nucleotide in the absence of GroES, only ATP was effective for reducing the affinity of GroEL for the target protein. However, when GroES was present, not only ATP but also the ATP analogs were found to effectively reduce the GroEL affinity for the target protein.
(2) We constructed E. coli expression systems for GFP and its Cycle3 mutant and studied their in vitro refolding reactions by fluorescence spectroscopy. These proteins can also be used as model target proteins of the chaperonin.
(3) We studied the interactions of GroEL with ADP and the ATP analog by titration calorimetry and fluorescence spectroscopy. In the latter method, we used the fluorescence intensity change of pyrenyl GroEL to monitor the interactions. We found that these nucleotides bound to GroEL in a non-cooperative manner and that there were two kinds of the binding sites with different affinities. Only ATP induced a cooperative fluorescence change of pyrenyl GroEL., suggesting that ATP hydrolysis was required for the cooperative change. From this results together with the above results in(1), the ATP hydrolysis and the resultant cooperative change are expected to by required for releasing the target protein form GroEL. Less

  • Research Products

    (35 results)

All Other

All Publications (35 results)

  • [Publications] Ikeguchi, M.: "Transition state in the folding of α-lactalbumin probed by the 6-120 disulfide bond"Protein Sci.. 7. 1564-1574 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mizuguchi, M.: "Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy"J. Mol. Biol.. 283. 265-277 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsurupa, G.P.: "Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL"J. Mol. Biol.. 277. 733-745 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Chaudhuri, T.K.: "Effect of the extra N-terminal methionine residue on the stability and folding of recombinant α-lactalbumin expressed in Escherichia coli"J. Mol. Biol.. 285. 1179-1194 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Fujiwara, K.: "Folding-unfolding equilibrium and kinetics of equine β-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate"Biochemistry. 38. 4455-4463 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Koshiba, T.: "Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein"Protein Eng.. 12. 429-435 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuwajima, K.: "Molten Globule"Encyclopedia of Molecular Biology (Creighton, T.E. ed) John Wiley & Sons, Inc. New York.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Maki, K.: "Effects of proline mutations on the folding of staphylococcal nuclease"Biochemistry. 38. 2213-2223 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Makio, T.: "Chaperonin-affected refolding of α-lactalbumin: Effects of nucleotides and the co-chaperonin GroES"J. Mol. Biol.. 293. 125-137 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Terada, T.P.: "Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding"Biochim. Biophys. Acta. 1431. 263-281 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桑島邦博: "シャペロニン:蛋白質立体構造形成の分子機械"パリティ. 14(12). 78-81 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Motojima, F.: "Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding"Biochem. Biophys. Res. Commun.. 267. 842-849 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Forge, V.: "Is folding of β-lactoglobulin non-hierarchic? Intermediate with native-like β-sheet and non-native α-helix"J. Mol. Biol.. 296. 1039-1051 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Arai, M.: "The role of the molten globule state in protein folding"Adv. Protein Chem.. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Arai, M.: "Effect of an Alternative Disulfide Bond on the Structure, Stability, and Folding of Human Lysozyme"Biochemistry. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ikura, T.: "Fast folding of Escherichia coli cylophilin A. A hypothesis of a unique hydrophobic core with a phenylalanine cluster"J. Mol. Biol.. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Koshiba, T.: "Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme"Biochemistry. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kuwajima, K.: "The molten globule state - the physical picture and biological significance"Mechanisms of protein folding 2nd ed. (Pain, R.H., ed.), Oxford University Press, Oxford. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ikeguchi, M. Fujino, M., Kato, M., Kuwajima, K. & Sugai, S.: "Transition state in the folding of α-lactalbumin probed by the 6-120 disulfide bond"Protein Sci. 7. 1564-1574 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mizuguchi, M., Arai, M., Ke, Y., Nitta, K. & Kuwajima, K.: "Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy"J. Mol. Biol.. 283. 265-277 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsurupa, G. P., Ikura, T., Makio, T. & Kuwajima, K.: "Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL"J. Mol. Biol.. 277. 733-745 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Chaudhuri, T. K., Horii, K., Yoda, T., Arai, M., Nagata, S., Terada, T. P., Uchiyama, H., Ikura, T., Tsumoto, K., Kataoka, H., Matsushima, M., Kuwajima, K. & Kumagai, I: "Effect of the extra N-terminal methionine residue on the stability and folding of recombinant α-lactalbumin expressed in Escherichia coli."J. Mol. Biol.. 285. 1179-1194 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Fujiwara, K., Arai, M., Shimizu, A., Ikeguchi, M., Kuwajima, K. & Sugai S.: "Folding-unfolding equilibrium and kinetics of equine β-lactoglobulin : equivalence between the equilibrium molten globule sate and a burst-phase folding intermediate."Biochemistry. 38. 4455-4463 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Koshiba, T., Hayashi, T., Miwako, I., Kumagai, I., Ikura, T., Kawano, K., Nitta, K. & Kuwajima, K.: "Expression of a synthetic gene encoding canine milk lysozyme in escherichia coli and characterization of the expressed protein."Protein Eng.. 12. 429-435 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Maki, K., Ikura, T., Hayano, T., Takahashi, N. & Kuwajima, K.: "Effects of proline mutations on the folding of staphylococcal nuclease."Biochemistry. 38. 2213-2223 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Makio, T., Arai, M. & Kuwajima, K.: "Chaperonin-affected refolding of α-lactalbumin : Effects of nucleotides and the co-chaperonin GroES."J. Mol. Biol.. 293. 125-137 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Terada, T. P. & Kuwajima, K.: "Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry : evidence for noncooperative nucleotide binding."Biochim. Biophys. Acta. 1431. 269-281 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Motojima, F., Makio, T., Aoki, K., Makino, Y., Kuwajima, K. & Yoshida, M.: "Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding."Biochem. Biophys. Res. Commun.. 267. 842-849 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Forge, V., Hoshino, M., Kuwata, K., Arai, M., Kuwajima, K., Batt, C. A. & Goto, Y.: "Is folding of β-lactoglobulin non-hierarchic? Intermediate with native-like β-sheet and non-native α-helix."J. Mol. Biol.. 296. 1039-1051 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Arai, M. & Kuwajima, K.: "The role of the molten globule sate in protein folding."Adv. Protein Chem.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Arai, M., Hamel, P., Kanaya, E., Inaka, K., Miki, K., Kikuchi, M. & Kuwajima, K.: "Effect of an Alternative Disulfide Bond on the Structure, Stability and Folding of Human Lysozyme."Biochemistry. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ikura, T., Hayano, T., Takahashi, N. & Kuwajima, K.: "Fast folding of Escherichia coli cylophilin A. A hypothesis of a unique hydrophobic core with a phenylalanine cluster."J. Mol. Biol.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Koshiba, T., Yao, M., Kobashigawa, Y., Demura, M., Nakagawa, A., Tanaka, I., Kuwajima, K. & Nitta, K.: "Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme."Biochemistry. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuwajima, K.: "John Wiley & Sons, Inc., New York."Molten Globule. In Encyclopedia of Molecular Biology (Creighton, T. E., ed.). 1531-1534 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kuwajima, K. & Arai, M.: "The molten globule state-the physical picture and biological significance. In Mechanisms of protein folding 2nd ed. (Pain, R. H., ed.)"Oxford University Press, Oxford (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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