title: A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin.
abstract: 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.
authors: Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
authors: Mio
authors: M
authors: Yamamoto
authors: Kanako
authors: K
authors: Nakagawa
authors: Kazuo
authors: K
authors: Fujiwara
authors: Akio
authors: A
authors: Shimizu
authors: Mitsunori
authors: M
authors: Ikeguchi
authors: Masamichi
authors: M
authors: Ikeguchi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D006736:Horses; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry; D011392:Proline; D017510:Protein Folding
chemical_list: D004220:Disulfides; D007782:Lactoglobulins; D010446:Peptide Fragments; D011392:Proline