Disulfide determinants of calcium-induced packing in alpha-lactalbumin.
alpha-Lactalbumin (alpha-LA) is a two-domain calcium-binding protein that folds through a molten globule intermediate. Calcium binding to the wild-type alpha-LA molten globule induces a transition to the native state. Here we assess the calcium-binding properties of the alpha-LA molten globule by studying two variants of alpha-LA. alpha-LA(alpha) contains only the two disulfide bonds in the alpha-helical domain of alpha-LA, while alpha-LA(beta) contains only the beta-sheet domain and interdomain disulfide bonds. We found that only alpha-LA(beta) binds calcium, leading to the cooperative formation of substantial tertiary interactions. In addition, the beta-sheet domain acquires a native-like backbone topology. Thus, specific interactions within alpha-LA imposed by the beta-sheet domain and interdomain disulfide bonds, as opposed to the two alpha-helical domain disulfides, are necessary for the calcium-induced progression from the molten globule toward more native-like structure. Our results suggest that organization of the beta-sheet domain, coupled with calcium binding, comprises the locking step in the folding of alpha-LA from the molten globule to the native state.
Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02142, USA.
L C
LC
Wu
B A
BA
Schulman
Z Y
ZY
Peng
P S
PS
Kim
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017433:Protein Structure, Secondary
D004220:Disulfides; D007768:Lactalbumin; D002118:Calcium