Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.
The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.
D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D011487:Protein Conformation; D009418:S100 Proteins / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*
D015415:Biomarkers; C030564:S-100 calcium-binding protein alpha subunit; D009418:S100 Proteins; D004492:Edetic Acid; D015032:Zinc; D002118:Calcium