Calcium binding peptides from alpha-lactalbumin: implications for protein folding and stability.
The calcium binding protein alpha-lactalbumin folds via a molten globule intermediate. Calcium does not bind strongly to the unfolded protein or the molten globule, but does bind to the transition state between the molten globule and the native protein. Of interest are the structures formed in the transition state that promote calcium binding. To study the importance of local secondary structure on calcium binding, we have synthesized two peptides corresponding to the calcium binding site that include the flanking C-helix and 3(10)-helix. The first peptide, elbow-A, consists of residues 72-100 from bovine alpha-lactalbumin, but with Cys 73, Cys 77, and Cys 91 replaced by alanines. In the second peptide, denoted elbow, the cysteines at position 73 and 91 are included and the nativelike disulfide bond is formed. Both peptides are monomeric and unstructured in aqueous solution and bind calcium weakly with apparent K(d)'s on the order of 10(-2) M. In 50% trifluoroethanol (v/v), the peptides are 45% helical as judged by CD. NMR studies performed on elbow and elbow-A in TFE indicate that the helical structure is confined to the C-helix. In this solvent system elbow binds calcium one-to-one with a K(d) of 50 microM. Removing the disulfide bond reduces, but does not eliminate calcium binding (K(d) = 170 microM in 50% TFE). These results suggest that formation of the C-helix promotes calcium binding and may be a key determinant of calcium binding in the transition state.
Kuhlman
B
B
Department of Chemistry, State University of New York at Stony Brook 11794-3400, USA.
Boice
J A
JA
Wu
W J
WJ
Fairman
R
R
Raleigh
D P
DP
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000494:pharmacology; D014461:Ultracentrifugation
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D014270:Trifluoroethanol; D007768:Lactalbumin; D002122:Calcium Chloride