Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli.
Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC 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 curves 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 Sbetagly 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 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.
Dipartimento di Chimica, Universit'a di Napoli Federico II, Napoli, Italy.
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F
Catanzano
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Graziano
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B
De Paola
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Barone
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D'Auria
M
M
Rossi
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Nucci
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D014364:Tryptophan; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014364:Tryptophan; D001617:beta-Glucosidase; D019791:Guanidine