Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a...

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C (m), occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T (m), (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.