Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin.
Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.
Ewbank
J J
JJ
European Molecular Biology Laboratory, Heidelberg, Germany.
Creighton
T E
TE
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005456:Fluorescent Dyes; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010084:Oxidation-Reduction; D017510:Protein Folding; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; C006421:1,5-I-AEDANS; D014508:Urea; D007768:Lactalbumin; D004492:Edetic Acid; D002118:Calcium; D004229:Dithiothreitol