Structural basis of the destabilization produced by an amino-terminal tag in the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus

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We have previously shown that the major ion-pairs network of the tetrameric β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the β-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85–95 °C and decreased the Tm of the protein of 6 °C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 °C whilst that of the wild type enzyme is stable up to 98 °C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the β-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.