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  • Title: Ca2+ and Zn2+ bind to different sites and induce different conformational changes in human calcyclin.
    Author: Kordowska J, Stafford WF, Wang CL.
    Journal: Eur J Biochem; 1998 Apr 01; 253(1):57-66. PubMed ID: 9578461.
    Abstract:
    Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately 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 Tyr fluorescence. Saturation occurs 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 Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations 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.
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