CHARACTERIZATION OF LANTHANIDE ION-BINDING TO THE EF-HAND PROTEIN S100-BETA BY LUMINESCENCE SPECTROSCOPY

S100 beta is a member of a group of low-molecular weight acidic calciu m binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and I I). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy us ing Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characte rize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta show s two distinct Eu3+ binding environments from both the excitation spec trum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd Of 660 +/- 20 nM, whereas the dissociation const ant for the pseudo-EF hand site I is significantly weaker. Lifetimes i n H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water mol ecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln(3+) binding domains I and II in c almodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta sh ow that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is si gnificantly different than that in the absence of Zn2+. Tyrosine-17-se nsitized Tb3+ luminescence experiments indicate that the Tb3+ occupyin g the proximal weaker binding site I is sensitized, whereas Tb3+ in si te II is not. The distance between sites I and II (15.0 +/- 0.4 Angstr om) in S100 beta was determined from Forster-type energy transfer in D 2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Z n2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 Angstro m. Location of histidine-15 close to pseudo-EF site I suggests that Zn 2+ binding likely changes the conformation of this site, causing a red uction of the intersite distance by approximately 2 Angstrom.