Multistate unfolding of α-mannosidase from Canavalia ensiformis (Jack Bean): Evidence for the thermostable molten globule

► Progressive appearance of class S, I, II and III trp conformers during unfolding of α-mannosidase. ► Multistate unfolding of the protein in presence of GdnHCl. ► Simultaneous dissociation, partial unfolding and aggregation of the protein during denaturation. ► Formation of a thermostable molten globule in the vicinity of 1 M GdnHCl. ► Earliest report on molten globule of the α-mannosidase from plant source. The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II α-mannosidase from Canavalia ensiformis (Jack Bean) (Jbα-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jbα-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jbα-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 °C and stability of the structure up to 80 °C unlike the native Jack Bean α-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II α-mannosidase.