No 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.

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine -lactoglobulin (ELG) was investigated by smallangle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/ C160A has a globular and compact conformation, like the wild type. 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. Funct. Bioinf. 63,[595][596][597][598][599][600][601][602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native -helix increases.