Table 1.

Thermodynamic parameters obtained from ITC measurements at 25°C for the binding of ERE duplex to the DB domain of ERα pre-treated with EDTA to strip divalent zinc ions (DB) and upon reconstitution with divalent ions of zinc (DB[Zn]), cadmium (DB[Cd]), mercury (DB[Hg]) and cobalt (DB[Co])

	Kd/nM	ΔH/kcal.mol−1	TΔS/kcal.mol−1	ΔG/kcal.mol−1	ΔCp/kcal.mol−1.K−1
DB	NB	NB	NB	NB	NB
DB[Zn]	68 ± 8	−25.20 ± 0.15	−15.40 ± 0.21	−9.80 ± 0.07	−0.28 ± 0.04
DB[Cd]	69 ± 5	−33.99 ± 0.25	−24.20 ± 0.20	−9.78 ± 0.04	−0.22 ± 0.04
DB[Hg]	59 ± 4	−39.95 ± 0.13	−30.07 ± 0.16	−9.88 ± 0.04	−0.85 ± 0.01
DB[Co]	81 ± 2	−29.35 ± 0.10	−19.66 ± 0.10	−9.69 ± 0.01	−0.60 ± 0.01

The values for the affinity (K_d) and enthalpy change (ΔH) accompanying the binding of ERE duplex to the DB domain reconstituted with various metals were obtained from the fit of a one-site model, based on the binding of a ligand to a macromolecule using the law of mass action, to the corresponding ITC isotherms as described earlier (Wiseman et al., 1989; Deegan et al., 2010). Free energy of binding (ΔG) was calculated from the relationship ΔG=RTlnK_d, where R is the universal molar gas constant (1.99 cal/mol/K) and T is the absolute temperature (K). Entropic contribution (TΔS) to binding was calculated from the relationship TΔS=ΔH−ΔG. Heat capacity change (ΔCp) was calculated from the slope of ΔH-T plot for the corresponding protein-DNA complex (Figure 2). Binding stoichiometries generally agreed to within ±10%. Errors were calculated from at least three independent measurements. All errors are given to one standard deviation. Note that the DB domain pre-treated with EDTA to strip divalent zinc ions and upon reconstitution with divalent metal ions of barium, copper, iron, lead, manganese, nickel and tin showed no binding (NB) to the ERE duplex.