Guanidine-induced denaturation of β-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli

Guanidine-induced denaturation of Sulfolobus solfataricus P-glycosidase expressed in Escherichia coli, S beta gly, was investigated at pH 6.5 and 25 degrees C by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL(-1). Moreover, the transition cut-yes determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL(-1) Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of S beta gly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degrees C and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, S beta gly has a stability similar to that of mesophilic proteins.