Unfolding analysis of the mature and unprocessed forms of Bacillus licheniformis γ-glutamyltranspeptidase

Abstract

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) undergoes an autocatalytic process to generate 44.9 and 21.7 kDa subunits; however, a mutant protein (T399A) loses completely the processing ability and mainly exists as a precursor. For a comprehensive understanding of their structural features, the biophysical properties of these two proteins were investigated by circular dichroism and fluorescence spectroscopy. Tryptophan fluorescence and circular dichroism spectra were nearly identical for BlGGT and T399A, but unfolding analyses revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and the unprocessed T399A displayed T(m) values of 61.4°C and 68.1°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Fluorescence quenching analysis showed that T399A had a dynamic quenching constant similar to that of the wild-type enzyme. BlGGT started to unfold beyond ∼2.14 M GdnHCl and reached an unfolded intermediate, [GdnHCl](0.5, N - U), at 2.85 M, corresponding to free energy change [Formula: see text] of 12.34 kcal mol( - 1), whereas the midpoint of the denaturation curve for T399A was approximately 3.94 M, corresponding to a [Formula: see text] of 4.45 kcal mol( - 1). Taken together, it can be concluded that the structural stability of BlGGT is superior to that of T399A.

Keywords: Autocatalytic processing; Bacillus licheniformis; Chemical denaturation; Thermal unfolding; γ-Glutamyltranspeptidase.

Fig. 1

Analysis of the purified enzymes by SDS-PAGE (a) and non-denaturing PAGE (b). The protein samples were electrophoresed on 12% polyacrylamide gel and visualized by Coomassie brilliant blue staining and activity staining. Lane M represents molecular size marker, and lanes 1 and 2 are BlGGT and T399A, respectively

Fig. 2

Intrinsic tryptophan fluorescence (a) and CD (b) spectra of BlGGT and T399A. An average of five spectra is shown for each protein was recorded at 25°C

Fig. 3

Unfolding and cooling curves of BlGGT and T399A in 20 mM Tris–HCl buffer (pH  8.0). Transitions were obtained by recording the ellipticity of the samples at 222 nm. Protein concentration was set to 1.5 mg mL^− 1

Fig. 4

Stern—Volmer plots for the quenching of BlGGT and T399A by acrylamide and Ni^2 + Prior to fluorescence analysis, the protein samples in 50 mM Tris-HCl buffer (pH 8.0) were incubated with the quenchers at room temperature for 30 min

Fig. 5

Unfolding curves of BlGGT and T399A in different concentrations of GdnHCl. Protein sample in 20 mM Tris—HCl buffer (pH  8.0) was incubated with various concentrations of GdnHCl for 30 min. The protein solution was then excited at 280 nm and the emission was recorded at 25°C. a AEW of these two proteins was used to analyze the unfolding data. b CD changes at 222 nm versus GdnHCl concentration