Energetics of structural domains in alpha-lactalbumin.
alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.
Hendrix
T M
TM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Griko
Y
Y
Privalov
P
P
D000818:Animals; D002151:Calorimetry; D002417:Cattle; D003553:Cystine / Q000737:chemistry; D004734:Energy Metabolism; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry*; D008892:Milk / Q000737:chemistry; D008958:Models, Molecular; D010215:Papio; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D003553:Cystine; D007768:Lactalbumin