(A) Urea melts of WT DBD as a function of buffered [Zn2+]free: 10−12 M (purple), 10−13 M (blue), 10−14M (yellow), 10−15 M (green), 10−16 M (red), 0 M (black). The m-values in kcal mol−1 M−1 of these fits are, in the same order: 3.1, 2.8, 2.6, 2.6, 1.9, and 3.0. (B) The relationship between ΔG and [Zn2+]free is similar whether or not DBD is holo or apo. (C) The Gibbs-Helmholtz equation relates the Gibbs free energy change of a process to the enthalpy change (ΔH), the temperature (T), and the change in heat capacity (ΔCp). (D) m-values for individual urea and guanidine melts as a function of temperature do not indicate two-state behavior. (E) Temperature dependence of apoDBD folding free energy, measured using guanidine denaturation and fit to the Gibbs-Helmholtz equation yields ΔHm = 160 ± 12 kcal mol−1, Tm = 301 ± 1 K, and ΔCp = 6.8 ± 1.1 kcal mol−1 K−1. For Gibbs-Helmholtz analysis, independent experimental data were pooled and fit once, and results are reported as the fit parameters and standard effort of the fit. Replicates otherwise consisted of independent experiments performed with the same preparations of purified proteins.