title: Tertiary structure in 7.9 M guanidinium chloride--the role of Glu53 and Asp287 in Pyrococcus furiosus endo-beta-1,3-glucanase.
abstract: The thermodynamic stability of family 16 endo-beta-1,3-glucanase(EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction,both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-beta-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 M guanidinium chloride. The persistence of tertiary structure in 7.9 M guanidinium chloride, a feature of the wild-type enzyme,is observed also for the three variant proteins. The DeltaG(H2O) values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximately 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ x mol(-1), suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 M guanidinium chloride comparable to that induced by the presence of calcium on the wildtype. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-beta-1,3-glucanase to thrive under adverse environmental conditions.
authors.affiliation: Dipartimento di Scienze Biochimiche, 'A. Rossi Fanelli' Sapienza Università di Roma, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: Rita
authors.initials: R
authors.lastname: Florio
authors.forename: Sebastiana
authors.initials: S
authors.lastname: Angelaccio
authors.forename: Giulio
authors.initials: G
authors.lastname: Gianese
authors.forename: Johan F T
authors.initials: JF
authors.lastname: van Lieshout
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.forename: Valerio
authors.initials: V
authors.lastname: Consalvi
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D020673:Protein Renaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D002614:Chelating Agents; D005456:Fluorescent Dyes; D001224:Aspartic Acid; D018698:Glutamic Acid; D004533:Egtazic Acid; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D019791:Guanidine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium