Site-selective laser spectroscopy of lanthanide-binding sites in calmodulin

Site-selective laser spectroscopy has been used to resolve the spectral features of lanthanide fluorescence probe ions in calcium-binding proteins. The capabilities and characteristics of this technique are studied using bovine brain calmodulin where the calcium-binding sites are very similar. Two distinct spectral features are identified. These features were followed during a Eu3+ titration and were found to fill successively, showing they correspond to the high- and low-affinity sites. One set of spectral features is assigned to domains I and III, which are the high-affinity domains, while the other set is assigned to domains II and IV. Additional nonspecific binding is observed after the domains are filled. Tb3+ titrations confirmed earlier results that the tyrosine-containing domains fill second and third (R. W. Wallace, E. A. Tallant, M. E. Duckter, and W. Y. Cheung, 1982, J. Biol. Chem. 257(4), 1845-1854). Site-selective laser spectroscopy was also used to identify the presence of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid contamination that could cause interference in titrations.