The Ca2+-binding sequence in bovine brain S100b protein beta-subunit. A spectroscopic study.
Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.
Baudier
J
J
Laboratoire de Physique, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France.
Cole
R D
RD
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure*; D002417:Cattle; D002942:Circular Dichroism; D011487:Protein Conformation; D009418:S100 Proteins / Q000378:metabolism* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013725:Terbium / Q000378:metabolism; D014443:Tyrosine; D015032:Zinc
D002135:Calcium-Binding Proteins; D009418:S100 Proteins; D013725:Terbium; D014443:Tyrosine; D015032:Zinc; D002118:Calcium