Two-dimensional IR correlation spectroscopy of mutants of the beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus identifies the mechanism of quaternary structure stabilization and unravels the sequence of thermal unfolding events

Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion-pairs compared to mesophilic glycoside hydrolases. The ion-pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. Here, the structure and thermal stability of the wild-type beta-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were studied by Fourier transform infrared spectroscopy. The IR data revealed small differences in the secondary structure of the proteins and showed a lower thermo stability of the mutant proteins with respect to the wild type. Generalized bidimensional correlation analysis of infrared spectra (2D-IR) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild type indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR correlation analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilises the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. These evidences give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophilic organisms.