Fig. 5. cop operator DNA binding activities of various C101A CopY metallostates. (A) Fluorescence anisotropy (FA)-based DNA binding experiment for Zn1 (black) and apo-states (red) of C101 CopY, with the continuous line a fit to 1 : 1 non-dissociable dimer-DNA binding model. Ka = 1.0 (±0.2) × 107 M–1 and Δr = 0.014 (±0.03) for Zn1 CopY and Ka = 2.8 (±0.6) × 106 M–1 and Δr = 0.017 (±0.03) for apo-CopY, for ΔGc = 0.6 (±0.1) kcal mol–1. Conditions: 0.23 M NaCl, 10 mM HEPES, pH 7.0, 2 mM TCEP, 40 μM ZnSO4 or 2 mM EDTA, 25 °C. (B) Representative electrophoretic mobility shift analysis (EMSA). Top, Zn1 C101 CopY dimer; middle, apo-state; bottom, Cu2 C101A CopY. B, bound; F, free DNA. The equilibrium Zn1 CopY-dimer DNA binding affinity, Ka, estimated from these data is 2.0 × 107 M–1, consistent with the FA-binding data in panel A. Solution conditions: 25 mM HEPES, pH 7.0, 200 mM NaCl (chelexed), 5 mM MgCl2, 50 μg mL–1 BSA, 25 °C. Apo-CopY samples contained 5 mM EDTA.
