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Papers In Press, published online ahead of print September 17, 2001
Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030
Corresponding Author: Rowen.Chang{at}uth.tmc.edu
The technique of disulfide scrambling permits reversible conversion of the native and denatured (scrambled) proteins via shuffling and reshuffling of disulfide bonds. Under strong denaturing conditions (e.g. 6M GdmCl) and in the presence of a thiol initiator, a-Lactalbumin (aLA) denatures by shuffling its four native disulfide bonds and converts to an assembly of 45 species of scrambled isomers. Among them, two predominant isomers, designated as X-aLA-a and X-aLA-d, account for about 50% of the total denatured structure of aLA. X-aLA-a and X-aLA-d, which adopt the disulfide patterns of [1-2,3-4,5-6,7-8] and [1-2,3-6,4-5,7-8] respectively, represent the most unfolded structures among the 104 possible scrambled isomers [Chang J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712]. In this study, X-aLA-a and X-aLA-d were purified and allowed to refold through disulfide scrambling to form the native aLA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase HPLC. The results revealed two major on-pathway productive intermediates, two major off-pathway kinetic traps and at least 30 additional minor transient intermediates. Of the two major on-pathway intermediates, one takes on a native-like a-helical domain and the other comprises a structured b-sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native like structures. Overall, the folding mechanism of aLA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.
J. Biol. Chem, 10.1074/jbc.M108057200
Submitted on August 21, 2001
Revised on September 14, 2001
Accepted on September 17, 2001
The folding pathway of a-Lactalbumin elucidated by the technique of disulfide scrambling isolation on-pathway and off-pathway intermediates
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