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. 2020 Dec 2;9:e61487. doi: 10.7554/eLife.61487

Figure 2. Zinc-binding affinity and stability of DBD.

(A) WT DBD (black) and full-length WT p53 (red) bind Zn2+ with KZn values of (1.6 ± 0.3) x 10−15 M and (0.4 ± 0.1) x 10−15 M, respectively, as determined by change in Tyr fluorescence (10°C, n = 3, SD). (B) Unfolded WT DBD (black) and unfolded C176S DBD (green), bind Zn2+ with KZn,U values of (42 ± 7) x 10−9 M and (50 ± 23) x 10−9 M, respectively, as determined by FluoZin-3 competition in 6 M urea (10°C, n = 3, SD). (C) Plotting folding free energy of DBD vs. [Zn2+]free (10°C) reveals that R175H (green) is a pure zinc-binding-class mutant whereas A138V (red) is a pure stability-class mutant. The point at which the lines deflect upwards are the approximate KZn values. WT DBD is in black. Open points denote outliers excluded from analysis. Outliers were identified on inspection and rejected if their exclusion (1) improved goodness of fit, and (2) produced a model for which they lay outside the 95% prediction interval. (D) Temperature dependence of apoDBD folding free energy fit to the Gibbs-Helmholtz equation (Figure 2—figure supplement 1C) yields ΔHm = 171 ± 20 kcal mol−1, Tm = 300 ± 1 K, and ΔCp = 7.0 ± 1.7 kcal mol−1 K−1 (fit value ± SE of fit). In (C and D), independent experimental data were pooled and fit once, and results are reported as the fit parameters and standard effort of the fit. Otherwise (A, B), replicates consisted of independent experiments performed with the same preparations of purified proteins, which were fit separately and the results pooled. Single curves are shown in the figure for illustration.

Figure 2.

Figure 2—figure supplement 1. Physical analysis validation data.

Figure 2—figure supplement 1.

(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.