Bimane- and acrylodan-labeled S100 proteins. Role of cysteines-85.alpha. and -84.beta. in the conformation and calcium binding properties of S100aa and S100b (.beta..beta.) proteins

Abstract

Bovine brain S100.alpha..alpha., S100a (.alpha..beta.), and S100b (.beta..beta.) protein dimers were labeled with the sulfhydryl-specific fluorescent probes monobromo(trimethylammonio)bimane (bimane) and 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) at cysteines-85.alpha. and -84.beta.. The conformation and fluorescence properties of the S100 proteins derived were studied by means of anion-exchange chromatography on a Mono Q column using a fast protein chromatography system and fluorescence intensity, maximum emission wavelength, and polarization measurements. Spectroscopic studies on the intrinsic absorption and fluorescence properties of S100.alpha..alpha. and S100b proteins chemically modified on cysteines-85.alpha. and -84.beta. with iodoacetamide completed this study. Several arguments suggest that the alkylated S100 proteins undergo conformational changes that are mainly characterized by the destablization of the quaternary protein structure, which provokes a slow dimer-monomer equilibrium at high protein concentrations and induces total subunit disssociation at low ones. Calcium binding studies on bimane-S100.alpha..alpha. and -S100b proteins showed that alkylated proteins had a much higher calcium binding affinity than native protein and that the antagonistic effect of KCl on calcium binding was much less pronounced. These results confirmed our previous observations that the affinities of calcium binding sites II.alpha. and II.beta. in S100 proteins are highly dependent on protein conformation [Baudier, J., and Gerard, D (1986) J. Biol. Chem. 261, 8204-8212].