Spectral studies on the calcium binding properties of bovine brain S-100b protein.

The effect of Ca2+ binding on the circular dichroism (CD) and 270-MHz proton nuclear magnetic resonance (NMR) spectra of brain-specific S-100b calcium binding protein has been examined at two pH values, 8.5 and 7.5. At pH 8.5, S-100b protein binds two Ca2+ per monomer with Kd values of 6 x 10(-5) and 2 x 10(-4) M, whereas at pH 7.5, the protein binds only one Ca2+ per monomer with a Kd of 2 x 10(-4) M. The presence of K+ inhibits the binding of Ca2+ to the higher affinity site at pH 8.5, and the affinity for calcium is lowered to Kd = 8.5 x 10(-4) M. Mg2+ has no effect on protein conformation. In the absence of Ca2+, S-100b undergoes a conformational change when the protein is titrated from pH 8.6 to 6.0. Addition of Ca2+ perturbed the environment of tyrosine and phenylalanine residues as measured by ultraviolet difference spectroscopy and 1H NMR. CD melt experiments and far-ultraviolet CD studies at alkaline pH and NMR experiments suggest that the protein is more stable in the presence of Ca2+. The single tyrosine residue in the protein ionizes only after the protein is denatured by exposure to high pH.