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The size and shape of S-100a and S-100b proteins in solution have been examined by gel filtration and ultracentrifugation in the presence and absence of Ca2+. S-100a and S-100b proteins, in the absence of Ca2+, have an intrinsic sedimentation coefficient, so20,w of 2.20 and 2.15 S, respectively and in 1 mM Ca2+ their so20,w values were decreased to 2.05 and 1.95 S, respectively, indicating an unfolding of the protein molecules. The Stokes radii of S-100a and S-100b (-Ca2+) were 23.4 A and 24.0 A and they decreased to 22.2 A and 22.3 A in the presence of Ca2+. The Ca2+ effect on S-100b greater than S-100a was in agreement with our earlier CD observations. Among the monovalent cations tested (K+, Na+ and Li+) K+ had the maximum effect on the Stokes radii and so20,w values of S-100 proteins. Since certain functions of the nervous system are accompanied by local changes in ionic concentrations of Ca2+, Na+ and K+, it is conceivable that these respective conformational changes induced in S-100 proteins by these metals may be related to their function in the brain.

The single cysteine on the alpha-subunit of bovine brain S-100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 +/- 2 nm, suggesting that the probe is in a fairly hydrophobic environment. Addition of Ca2+ to the protein caused the emission maximum to undergo a red shift to 504 +/- 2 nm, implying that the fluorophore is now more exposed to the solvent. Zn2+, when added to the protein, induced only a small perturbation and the emission maximum shifted to 481 +/- 2 nm. Ca2+ was able to perturb the fluorophore in the presence of Zn2+. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS)-labelled alpha-subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca2+. Addition of Ca2+ resulted in an increase in TNS fluorescence accompanied by a blue shift of the emission maximum to 445 +/- 1 nm indicating that the probe in the presence of Ca2+ moves to a hydrophobic domain. The fact that Ca2+ and Zn2+ can perturb the labelled sulfhydryl group in the presence of each other clearly demonstrates that the binding sites for the two metal ions must be different on the alpha-subunit as well as on the S-100a protein.