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

Abstract

The effect of Ca2+ binding on the circular dichroism (CD) and 270 MHz proton NMR spectra of brain-specific S-100b Ca binding protein was examined at pH 8.5 and 7.5. At pH 8.5, S-100b protein binds 2 Ca2+/monomer with Kd values of 6 .times. 10-5 and 2 .times. 10-4, whereas at pH 7.5, the protein binds only 1 Ca2+/monomer with a Kd of 2 .times. 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 Ca is lowered to Kd = 8.5 .times. 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 UV difference spectroscopy and 1H NMR. CD melt experiments and far-UV 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.