Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine β‐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.

Chloride-ion concentration dependence of molecular dimension in the acid-denatured state of equine β-lactoglobulin

“…C66A/C160A was expressed and purified as previously described (Yamada et al, 2006). Chemicals were of analytical grade from Nacalai Tesque or Wako Pure Chemical Industries (Osaka, Japan).…”

“…In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al (2006). Proteins Struct.…”

“…In the case of ELG, the chain expansion and concomitant increase of the helical content were expected at a low pH and low ionic strength. It has been found, however, that the helix formation does not occur even at a low ionic strength (Yamada et al, 2006). This may be due to the presence of disulfide bonds.…”

“…Previously, we constructed a mutant in which Cys66 and Cys160 were replaced with alanine (C66A/C160A) and the chloride-ion concentration dependence of its CD spectrum was measured at acidic pH. The CD spectrum of C66A/ C160A did show the increased helix content at a low ionic strength (Yamada et al, 2006). In this study, thus, we have investigated the chloride-ion concentration dependence of the molecular shape of C66A/C160A and wild-type ELG (WT) at acidic pH.…”

Structure and stability of Gyuba, a β‐lactoglobulin chimera

“…36 Refolded Gyuba was purified by DEAESepharose chromatography with a linear gradient of 0-350 mM NaCl in 50 mM Tris-HCl (pH ¼ 8.0). The fraction that contained Gyuba was further purified by gel-filtration chromatography (Sephacryl S-100 equilibrated with 5 mM NH 4 HCO 3 ) and then by QSepharose chromatography with a linear gradient of 0-500 mM NaCl in 20 mM PIPES (pH ¼ 7.0).…”