Locating the metal ion in calcium-binding proteins by using cerium(III) asa probe

The detection and assignment of NMR spectroscopic signals of carbon atoms f rom carbonyl and carboxylate groups in the loop hosting the Ce-III ion was performed for the cerium-substituted calcium-binding protein calbindin D-9k . This provided a tool to characterize in solution the first coordination s phere of the metal ion. Due to the well-documented possibility of replacing calcium with metal ions of the Ln(III) series, this approach turns out to be extremely efficient for characterizing in solution the coordination of c alcium ions in proteins, independently of, the availability of X-ray crysta l structures. The present approach completes the structural characterizatio n of lanthanide-substituted calcium-binding proteins, for which the role of long-range constraints arising from hyperflne interaction and self-orienta tion has already been assessed.