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Dynamic fluorescence studies of β‐glycosidase mutants from Sulfolobus solfataricus : Effects of single mutations on protein thermostability
Author(s) -
Bismuto Ettore,
Febbraio Ferdinando,
Limongelli Simona,
Briante Raffaella,
Nucci Roberto
Publication year - 2003
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10317
Subject(s) - sulfolobus solfataricus , thermostability , glycoside hydrolase , biochemistry , hydrolase , hyperthermophile , sulfolobus , pyrococcus furiosus , archaea , chemistry , protein structure , biology , enzyme , gene
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. Proteins 2003;51:10–20. © 2003 Wiley‐Liss, Inc.