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Tertiary structure in 7.9 m guanidinium chloride - the role of Glu53 and Asp287 in Pyrococcus furiosus endo-β-1,3-glucanase

Chiaraluce, Roberta, Florio, Rita, Angelaccio, Sebastiana, Gianese, Giulio, van Lieshout, Johan F.T., van der Oost, John, Consalvi, Valerio
FEBS journal 2007 v.274 no.23 pp. 6167-6179
Pyrococcus furiosus, alanine, binding sites, calcium, carboxylic acids, chelating agents, energy, environmental factors, guanidinium, mutants, mutation, prediction, proteins, surface area, thermodynamics, titration
The thermodynamic stability of family 16 endo-β-1,3-glucanase (EC 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-β-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 ΔGH²O 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-β-1,3-glucanase to thrive under adverse environmental conditions.