Bimane- and acrylodan-labeled S100 proteins. Role of cysteines-85 alpha and -84 beta in the conformation and calcium binding properties of S100 alpha alpha and S100b (beta beta) proteins.
Bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein dimers were labeled with the sulfydryl-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 destabilization of the quaternary protein structure, which provokes a slow dimer-monomer equilibrium at high protein concentrations and induces total subunit dissociation 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., & Gerard, D. (1986) J. Biol. Chem. 261, 8204-8212].
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D009281:Naphthalenes / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D009418:S100 Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds; D005456:Fluorescent Dyes; D009281:Naphthalenes; D009418:S100 Proteins; C021149:bimanes; C038518:acrylodan; D015081:2-Naphthylamine; D003545:Cysteine; D002118:Calcium