Is folding of β-Lactoglobulin non-hierarchic?: Intermediate with native-like β-sheet and non-native α-helix

The refolding of beta-lactoglobulin, a beta-barrel protein consisting of beta strands beta A-beta I and one major helix, is unusual because non-native alpha-helices are formed at the beginning of the process. We studied the refolding kinetics of bovine beta-lactoglobulin A at pH 3 using the stopped-now circular dichroism and manual H/H-2 exchange pulse labeling coupled with heteronuclear NMR. The protection pattern from the H/H-2 exchange of the native state indicated the presence of a stable hydrophobic core consisting of beta F, beta G and beta H strands. The protection pattern of the kinetic intermediate obtained about one second after initiating the reaction was compared with that of the native state. In this relatively late kinetic intermediate, which still contains some non-native helical structure, the disulfide-bonded beta-hairpin made up of beta G and beta H strands was formed, but the rest of the molecule was fluctuating, where the non-native alpha-helices may reside. Subsequently, the core beta-sheet extends, accompanied by a further alpha-helix to beta-sheet transition. Thus, the refolding of beta-lactoglobulin exhibits two elements: the critical role of the core beta-sheet is consistent with the hierarchic mechanism, whereas the alpha-helix to beta-sheet transition suggests the non-hierarchic mechanism. (C) 2000 Academic Press.