Calcium-induced tertiary structure modifications of endo-B-1,3-glucanase form Pyrococcus furiosus in 7.9 M guanidinium chloride

R. Chiaraluce, G. Gianese, S. Angelaccio, R. Florio, J.F.T. van Lieshout, J. van der Oost, V. Consalvi

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)

Abstract

The family 16 endo-b-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-b-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases
Original languageEnglish
Pages (from-to)515-524
JournalBiochemical Journal
Volume386
Issue number3
DOIs
Publication statusPublished - 2005

Keywords

  • transform infrared-spectroscopy
  • protein secondary structure
  • 3-dimensional structures
  • ftir spectroscopy
  • free-energy
  • recognition
  • database
  • endo-beta-1,3-glucanase
  • intermediate
  • alignments

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