THE BURST-PHASE INTERMEDIATE IN THE REFOLDING OF BETA-LACTOGLOBULIN STUDIED BY STOPPED-FLOW CIRCULAR-DICHROISM AND ABSORPTION-SPECTROSCOPY

The kinetics of the guanidine hydrochloride-induced unfolding and refo lding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichr oism and absorption measurements at pH 3.2 and 4.5 degrees C. The refo lding reaction was a complex process composed of different kinetic pha ses, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dea d time of stopped-flow measurements (similar to 18 ms), showed more in tense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and it s disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermedia te, apparently suggesting that the folding of beta-lactoglobulin is no t represented by a simple sequential mechanism. (2) The burst-phase in termediate has, however, a number of properties in common with the fol ding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the s equential model. These properties include: the presence of the seconda ry structure without the specific tertiary structure; formation of hyd rophobic core; broad unfolding transition of the intermediate; and rap idity of formation of the intermediate. The burst-phase intermediate o f beta-lactoglobulin is thus classified as the same species as the mol ten globule state. (3) The circular dichroism spectra of beta-lactoglo bulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochl oride suggests the presence of the residual beta-structure in the unfo lded state and the stabilization of the beta-structure by disulfide bo nds. Thus, if this residual P-structure is part of the native beta-str ucture and forms a folding initiation site, the folding reaction of be ta-lactoglobulin may not necessarily be inconsistent with the sequenti al model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part. (C) 1996 Academic Pre ss Limited