title: Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization.
abstract: Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssbeta-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles.
authors.affiliation: Institute of Protein Biochemistry and Enzymology, CNR, Naples, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Cobucci-Ponzano
authors.forename: Marco
authors.initials: M
authors.lastname: Moracci
authors.forename: Barbara
authors.initials: B
authors.lastname: Di Lauro
authors.forename: Maria
authors.initials: M
authors.lastname: Ciaramella
authors.forename: Rossana
authors.initials: R
authors.lastname: D'Avino
authors.forename: Mosé
authors.initials: M
authors.lastname: Rossi
mesh_terms: D004795:Enzyme Stability; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013696:Temperature
chemical_list: D007477:Ions; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase