The folding pathway of α-lactalbumin elucidated by the technique of disulfide scrambling -: Isolation of on-pathway and off-pathway intermediates

被引:32
作者
Chang, JY [1 ]
机构
[1] Univ Texas, Inst Mol Med, Res Ctr Prot Chem, Houston, TX 77030 USA
[2] Univ Texas, Dept Biochem & Mol Biol, Houston, TX 77030 USA
关键词
D O I
10.1074/jbc.M108057200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, a-lactalbumin (alphaLA) 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-alphaLA-a and X-alphaLA-d, account for about 50% of the total denatured structure of alphaLA. X-alphaLA-a and X-alphaLA-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-alphaLA-a and X-alphaLA-d were purified and allowed to refold through disulfide scrambling to form the native alphaLA. 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 a-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 alphaLA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.
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页码:120 / 126
页数:7
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