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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

The thermodynamic stability of family 16 endo‐β‐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‐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.