13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

Abstract

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca 2+ in both the CD and EF calcium binding sites, with Cd 2+ in both sites, with 113Cd 2+ in both sites, and with 113Cd 2+ in the CD site and Lu 3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C- 113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd 2+ from the EF site by Lu 3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd 2+/Lu 3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.