Tertiary structure in 7.9 M guanidinium chloride: the role of Glu-53 and Asp-287 in Pyrococcus furiosus endo-beta-1,3-glucanase

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

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Scopus)

Abstract

The thermodynamic stability of family 16 endo-ß-1,3-glucanase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-ß-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 m guanidinium chloride. The persistence of tertiary structure in 7.9 m guanidinium chloride, a feature of the wild-type enzyme, is observed also for the three variant proteins. The ¿GH2O values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximatelty 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ·mol¿1, suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent-exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 m guanidinium chloride comparable to that induced by the presence of calcium on the wild-type. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-ß-1,3-glucanase to thrive under adverse environmental conditions.
Original languageEnglish
Pages (from-to)6167-6179
JournalFEBS Journal
Volume274
DOIs
Publication statusPublished - 2007

Keywords

  • denatured state
  • staphylococcal nuclease
  • protein stability
  • unfolded state
  • thermophilic proteins
  • residual structure
  • m-values
  • intermediate
  • thermodynamics
  • salt

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