Characterization of lanthanide(III) ion binding to calmodulin using luminescence spectroscopy

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Abstract
Pulsed dye laser excitation spectroscopy of the 7F0 .fwdarw. 5D0 transition of Eu(III) reveals only a single peak as this ion is titrated into apocalmodulin. A titration based on the intensity of this transition shows that the first two Eu(III) ions bind quantitatively to two tight sites, followed by weaker binding (Kd = 2 .mu.M) to two additional sites under conditions of high ionic strength (0.5 M KCl). This excitation experiment is also shown to be a general method for measuring contaminating levels of EDTA down to 0.2 .mu.M in proton solutions. Experiments with Tb(III) using both direct laser excitation and indirect sensitization of Tb(III) luminescence through tyrosine residues in calmodulin also give evidence for two tight and two weaker binding sites (Kd = 2-3 .mu.M). The indirect sensitization results primarily upon binding to the two weaker sites, implying that Tb(III) binds first to domains I and II, which are remote from tyrosine-containing domains III and IV. The 7F0 .fwdarw. 5D0 excitation signal of Eu(III) was used to measure the relative overall affinities of the tripositive lanthanide ions, Ln(III), across the series. Ln(III) ions at the end of the series are found to bind more weakly than those at the beginning and middle of the series. Eu(III) excited-state lifetime measurements in H2O and D2O reveal that two water molecules are coordinated to the Eu(III) at each of the four metal ion binding sites. Measurements of Forster-type nonradiative energy-transfer efficiencies between Eu(III) and Nd(III) in the two tight sites were carried out by monitoring the excited-state lifetimes of Eu(III) in the presence and absence of the energy acceptor ion Nd(III). Measured energy-transfer efficiencies of 0.126 and 0.396 were obtained in H2O and D2O solutions, respectively. By use of a spectral overlap integral obtained from the absorption spectrum of Nd(III) bound in the two tight sites of calmodulin and a corrected emission spectrum for Eu(III) in calmodulin, a distance estimation of 12.1 .+-. 0.5 .ANG. was obtained, with the H2O and D2O results agreeing to within 0.2 .ANG.. This result supports a postulated structure for calmodulin based on its homology with parvalbumin.
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