The Ca2+-binding sequence in bovine brain S100b protein β-subunit. A spectroscopic study

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Abstract
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+ seqentially 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.cntdot.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.cntdot.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.cntdot.Zn2+6 complex.