Pathway of disulfide-coupled unfolding and refolding of bovine alpha-lactalbumin.
alpha-Lactalbumin's four disulfide bonds have been used to probe the nature of its native, molten globule, and unfolded states. The Ca2+ concentration could be used to vary the stability of the native state because it binds a single calcium ion with high affinity. In the Ca(2+)-bound native state, the Cys6-120 and Cys28-111 disulfides were reduced sequentially, reflecting their relative accessibilities. The native disulfides do not stabilize the native conformation as much as in other proteins, nor was the unfolding process as cooperative. After two disulfides were reduced, only part of the protein molecule appeared to remain folded. In the absence of Ca2+, the native state was much less stable, and a molten globule-type conformation tended to be adopted. Formation of disulfide bonds in the reduced protein was initially noncooperative and nonspecific. The molten globule state increased the rate at which disulfides were formed, by tending to keep close in space cysteine residues that are distant in the sequence. A large number of nonnative disulfide pairings were preferred, and the disulfides were not stabilized to any great extent. The alpha-lactalbumin molten globule seems to be stabilized by nonspecific interactions and without a well-defined topology. The native protein was regenerated only when Ca2+ bound to and stabilized the three-disulfide intermediate lacking the Cys6-120 disulfide, which then rapidly formed the fourth disulfide bond. The rate-limiting intramolecular steps were rearrangement of disulfide bonds to native combinations in species with two and three disulfide bonds.
Ewbank
J J
JJ
European Molecular Biology Laboratory, Heidelberg, Germany.
Creighton
T E
TE
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D013816:Thermodynamics
D004220:Disulfides; D007768:Lactalbumin; D004492:Edetic Acid; D005978:Glutathione; D002118:Calcium; D004229:Dithiothreitol