Dynamic fluorescence studies of β-glycosidase mutants from Sulfolobus solfataricus: Effects of single mutations on protein thermostability

Abstract

Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of β-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two α-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of β-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic β-glycosidases, in the thermal adaptation of archaea β-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. © 2003 Wiley-Liss, Inc.