Ca2+, K+‐Regulated Intramolecular Crosslinking of S‐100 Protein via Disulfide Bond Formation

Abstract

Reaction of the thiol reagent 5,5''-dithio-bis(2-nitrobenzoic acid) (Nbs2) with the brain-specific protein S-100 favored stabilization of the quaternary structure of the protein via disulfide bond formation. This process was modulated by those cations (Ca2+ and K+) known to affect the conformation of the protein. Ca2+ markedly favored the reaction of S-100 with Nbs2 but inhibited subsequent disulfide bond formation; K+ was much less effective in promoting interaction with Nbs2 but strongly stimulated disulfide bond formation. These findings are interpreted assuming that in presence of Ca2+ the 3 subunits forming the native S-100 protein have 2 cysteine residues exposed to the solvent but mismatched to form disulfides while in presence of K+ the sulfhydryl groups are in a less accessible position to Nbs2 but suitable for S-S bond formation. Crosslinking of S-100 subunits was characterized by the appearance in dodecylsulfate electrophoresis of 2 very close protein bands having a molecular weight almost identical to that of the native, undenatured protein but not of higher or lower-molecular-weight components. This finding, and the demonstration that the crosslinked and native S-100 proteins have identical profiles when analyzed by sucrose density centrifugation or gel chromatography indicate that disulfide bond formation occurs among subunits of the same molecule.