CA2-CHANGES IN HUMAN CALCYCLIN( AND ZN2+ BIND TO DIFFERENT SITES AND INDUCE DIFFERENT CONFORMATIONAL)

Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in E scherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect t o the oxidation state of the single Cys residue at position 3. CaCY wi th the SH group either reduced, blocked or oxidized stays as a dimer a s shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Sr fluorescence, the apparent binding constant (K-a) being 2.8-5.8X10(4)M(-1). Oxidized CaCY binds Ca2+ appr oximately twice as weakly than its reduced form. The affinity for Ca2 is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0X10(7)M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Sr fluorescence. Saturation occ urs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Z n2+ the fluorescence changes accompanying the binding, and the CaCY re activity with a hydrophobic probe, it was concluded that the two catio ns bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.