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Molecular Biology

, Volume 52, Issue 1, pp 69–74 | Cite as

Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro

  • V. V. Marchenkov
  • N. V. Kotova
  • T. A. Muranova
  • G. V. Semisotnov
Structural Functional Analysis of Biopolymers and Their Complexes

Abstract

GroES is a heptameric partner of tetradecameric molecular chaperone GroEL, which ensures the correct folding and assembly of numerous cellular proteins both in vitro and in vivo. This work demonstrates the results of a study of structural aspects of GroES that affect its interaction with GroEL and reassembly. The effect of limited trypsinolysis of GroES on these processes has been studied. It has been shown that limited trypsinolysis of GroES is only strongly pronounced outside the complex with GroEL and results in the cleavage of the peptide bond between Lys20 and Ser21. The N-terminal fragment (~2 kDa) is retained in the GroES particle, which maintains its heptaoligomeric structure but loses the ability to interact with GroEL and dissociates upon a change in the pH from 7 to 8. Trypsin-nicked GroES cannot reassemble after urea-induced unfolding, while the urea-induced unfolding of intact GroES is fully reversible. The reported results indicate the important role of the N-terminal part of GroES subunit in the assembly of its heptameric structure and the interaction with GroEL.

Keywords

oligomeric protein assembly GroES chaperonin limited trypsinolysis 

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References

  1. 1.
    Ellis R.J. 1999. Molecular chaperones: Pathways and networks. Curr. Biol. 9, R137–R139.CrossRefPubMedGoogle Scholar
  2. 2.
    Frydman J., Hartl F.U. 1996. Principles of chaperoneassisted protein folding: Differences between in vitro and in vivo mechanisms. Science. 272, 1497–1502.CrossRefPubMedGoogle Scholar
  3. 3.
    Martin J., Horwich A.L., Hartl F.U. 1992. Prevention of protein denaturation under heat stress by the chaperonin Hsp60. Science. 258, 995–998.CrossRefPubMedGoogle Scholar
  4. 4.
    Bergeron J.J., Craig E.A., Horwich A.L., et al. 1997. Molecular chaperones in biology and medicine at Obernai. Cell Stress Chaperones. 2, 220–228.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Roseman A.M., Chen S., White H., et al. 1996. The chaperonin ATPase cycle: Mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell. 87, 241–251.CrossRefPubMedGoogle Scholar
  6. 6.
    Yebenes H., Mesa P., Munoz I.G., et al. 2011. Chaperonins: Two rings for folding. Trends Biochem. Sci. }}36}}, 424–432.Google Scholar
  7. 7.
    Chen S., Roseman A.M., Hunter A.S., et al. 1994. Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. Nature. 371, 261–264.CrossRefPubMedGoogle Scholar
  8. 8.
    Fenton W. A, Kashi Y., Furtak K., et al. 1994. Residues in chaperonin GroEL required for polypeptide binding and release. Nature. 371, 614–619.CrossRefPubMedGoogle Scholar
  9. 9.
    Hunt J.F., Weaver A.J., Landry S.J., et al. 1996. The crystal structure of the GroES co-chaperonin at 2.8 Å resolution. Nature. 379, 37–45.CrossRefPubMedGoogle Scholar
  10. 10.
    Langer T., Pfeifer G., Martin J., et al. 1992. Chaperoninmediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11, 4757–4765.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Xu Z., Horwich A.L., Sigler P.B. 1997. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 388, 741–750.CrossRefPubMedGoogle Scholar
  12. 12.
    Marchenkov V.V., Semisotnov G.V. 2009. GroEL-assisted protein folding: Does it occur within the chaperonin inner cavity? Int. J. Mol. Sci. 10, 2066–2083.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Todd M.J., Viitanen P.V., Lorimer G.H. 1994. Dynamics of the chaperonin ATPase cycle: Implications for facilitated protein folding. Science. 265, 659–666.CrossRefPubMedGoogle Scholar
  14. 14.
    Seale J.W., Horowitz P.M. 1995. The C-terminal sequence of the chaperonin GroES is required for oligomerization. J. Biol. Chem. 270, 30268–30270.CrossRefPubMedGoogle Scholar
  15. 15.
    Lissin N.M., Venyaminov S.Y., Girshovich A.S. 1990. (Mg-ATP)-dependent self-assembly of molecular chaperone GroEL. Nature. 348, 339–342.CrossRefPubMedGoogle Scholar
  16. 16.
    Ryabova N., Marchenkov V., Kotova N., Semisotnov G. 2014. Chaperonin GroEL reassembly: an effect of protein ligands and solvent composition. Biomolecules. 4, 458–473.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Goldenberg D.P., Creighton T.E. 1984. Gel electrophoresis in studies of protein conformation and folding. Anal. Biochem. 138, 1–18.CrossRefPubMedGoogle Scholar
  18. 18.
    Guex N., Peitsch M.C. 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis. 18, 2714–2723.CrossRefPubMedGoogle Scholar
  19. 19.
    Landry S.J., Zeilstra-Ryalls J., Fayet O., et al. 1993. Characterization of a functionally important mobile domain of GroES. Nature. 364, 255–258.CrossRefPubMedGoogle Scholar
  20. 20.
    Llorca O., Schneider K., Carrascosa J.L., et al. 1997. Role of the amino terminal domain in GroES oligomerization. Biochim. Biophys. Acta. 1337, 47–56.CrossRefPubMedGoogle Scholar
  21. 21.
    Seale J.W., Gorovits B.M., Ybarra J., et al. 1996. Reversible oligomerization and denaturation of the chaperonin GroES. Biochemistry. 35, 4079–4083.CrossRefPubMedGoogle Scholar
  22. 22.
    Boudker O., Todd M.J., Freire E. 1997. The structural stability of the co-chaperonin GroES. J. Mol. Biol. 272, 770–779.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • V. V. Marchenkov
    • 1
  • N. V. Kotova
    • 1
  • T. A. Muranova
    • 2
  • G. V. Semisotnov
    • 1
  1. 1.Institute of Protein ResearchRussian Academy of SciencesPushchino, Moscow oblastRussia
  2. 2.Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesPushchino, Moscow oblastRussia

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