Ion-binding properties of recombinant S100β and two derivatives with either an inactivated Ca²⁺ site II or a normalized Ca²⁺ site I

S100β contains one unusual and one canonical Ca²⁺-binding motif. In this study, we measured Ca²⁺-binding and ensuing conformational changes of recombinant S100β (rS100β) and of two mutant forms in which either the canonical loop was inactivated (NoEF) or the unusual one replaced by a canonical one (Caloops). Caloops binds two Ca²⁺ per monomer with a 3-fold higher affinity than rS100β; the affinity of NoEF was too low for accurate direct determination. All three proteins bind 3-4 Zn²⁺ per monomer. Tyrosine 17 fluorescence spectra showed a decrease of intensity upon binding of Ca²⁺ to the three proteins and an increase upon binding of Zn²⁺ to rS100β and NoEF but not in Caloops. The fluorescence change as a function of the Ca²⁺ concentration yielded half-maximal changes ([Ca²⁺]₀₅) at 1.7, 11.3 and 0.55 mM free Ca²⁺ for rS100β, NoEF and Caloops, respectively. Our data demonstrate that in S100β alterations in one site can affect the Ca²⁺ binding properties of the other site.