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Originally published In Press as doi:10.1074/jbc.M108057200 on September 17, 2001

J. Biol. Chem., Vol. 277, Issue 1, 120-126, January 4, 2002
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The Folding Pathway of alpha -Lactalbumin Elucidated by the Technique of Disulfide Scrambling
ISOLATION OF ON-PATHWAY AND OFF-PATHWAY INTERMEDIATES*

Jui-Yoa ChangDagger

From the Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030

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. 6 M guanidinium chloride) and in the presence of a thiol initiator, alpha -lactalbumin (alpha LA) 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-alpha LA-a and X-alpha LA-d, account for about 50% of the total denatured structure of alpha LA. X-alpha LA-a and X-alpha LA-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-alpha LA-a and X-alpha LA-d were purified and allowed to refold through disulfide scrambling to form the native alpha LA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase high pressure liquid chromatography. 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 alpha -helical domain, and the other comprises a structured beta -sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native-like structures. Overall, the folding mechanism of alpha LA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.

* This work was supported by an endowment from the Robert Welch foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Inst. of Molecular Medicine, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-500-2458; Fax: 713-500-2424; E-mail: Rowen.Chang@uth.tmc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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