Equine β-lactoglobulin (ELG) assumes non-native
helices during
refolding and in partially folded states. Previously, circular dichroism
(CD) combined with site-directed mutagenesis identified helical regions
in the acid- and cold-denatured states of ELG. It is also known that
a fragment of ELG, CHIBL (residues 88–142), has a structure
similar to that of the cold-denatured state. For the study reported
herein, the structure of a shorter fragment, CHIBLΔF (residues
97–142), was investigated by CD and nuclear magnetic resonance
spectroscopy. The secondary chemical shifts clearly showed that non-native
α-helices are present in two different regions, residues 98–107
and 114–135, and are connected by a native
disulfide bond. The CD spectra of two peptides that correspond to
the helical regions are characterized by weak helical signatures,
and the sum of their CD spectra is nearly the same as the spectrum
of disulfide-reduced CHIBLΔF. Therefore, the non-native helices
are stabilized by the disulfide, and non-native helix formation may
occur only during the refolding of the disulfide-intact protein. Supporting
this conclusion is the observation that tear lipocalin, a homologue
of ELG that lacks the disulfide, does not form non-native helices
during folding.