title: Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin.
abstract: A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.
authors.affiliation: Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Yamada
authors.forename: Kanako
authors.initials: K
authors.lastname: Nakagawa
authors.forename: Takeo
authors.initials: T
authors.lastname: Yajima
authors.forename: Keiko
authors.initials: K
authors.lastname: Saito
authors.forename: Akihito
authors.initials: A
authors.lastname: Tokushima
authors.forename: Kazuo
authors.initials: K
authors.lastname: Fujiwara
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ikeguchi
mesh_terms: D000818:Animals; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry*; D006736:Horses; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011489:Protein Denaturation / Q000235:genetics; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D007782:Lactoglobulins