Unfolding and Refolding Pathways of a Major Kinetic Trap in the Oxidative Folding of α-Lactalbumin

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Abstract
α-Lactalbumin (αLA)-IIIA is a major kinetic intermediate present along the pathways of reductive unfolding and oxidative folding of bovine α-lactalbumin (αLA). It is a three-disulfide variant of native αLA lacking Cys6−Cys120 at the α-helical domain. Stability and the unfolding/refolding mechanism of carboxymethylated αLA-IIIA have been investigated previously by stop-flow circular dichroism (CD) and fluorescence spectroscopy. A stable intermediate compatible with molten globule was shown to exist along the pathways of unfolding-refolding of αLA-IIIA [Ikeguchi et al. (1992) Biochemistry 31, 16695−12700; Horng et al. (2003) Proteins 52, 193−202]. We investigate here the unfolding−refolding pathways and conformational stability of αLA-IIIA using the method of disulfide scrambling with the following specific aims: (a) to isolate and characterize the observed stable molten globule, (b) to analyze the heterogeneity of folding−unfolding intermediates, (c) to elucidate the disulfide structure of extensively unfolded isomer of αLA-IIIA, and (d) to clarify the relative conformational stability between αLA-IIIA and αLA. Two scrambled isomers, designated as X-αLA-IIIA-c and X-αLA-IIIA-a (X stands for scrambled), were isolated under mild and strong denaturing conditions. Their disulfide structures, CD spectra, and manners of refolding to form the native αLA-IIIA were analyzed in this report. The results are consistent with the notion that X-αLA-IIIA-c and X-αLA-IIIA-a represent a partially unfolded and an extensively unfolded isomers of native αLA-IIIA, respectively. The unfolding−refolding pathways of αLA-IIIA are elaborated and compared with that of intact αLA. These results display new insight into one of the most extensively studied molecules in the field of protein folding and unfolding.