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. 2016 Mar 14;5:e13909. doi: 10.7554/eLife.13909

Figure 4. The closed dimeric conformation of TAp63α constitutes a kinetically trapped state.

(A) TAp63αmin samples were incubated for 1 hr at different urea concentrations and subjected to size exclusion chromatography (SEC) at corresponding urea concentrations. (B) TAp63αmin samples were incubated in 1.75 M urea and injected into a Superose 6 3.2/300 column equilibrated with 1.75 M urea at different time points. (C) SEC profiles of TAp63αmin injected after incubation for 50 min in 1.75 M urea. Fractions of tetrameric and dimeric protein are highlighted in orange and blue, respectively. (D,E) SEC profiles of reinjected tetrameric (E) and dimeric (D) fractions (originating from SEC shown in C) after dialysis to 0 M urea for 13 hr. (F,G) SEC-MALS of TAp63αmin at different urea concentrations to proof the tetrameric nature of the early eluting peak in A. a, t and d denote aggregate, tetramer and dimer respectively. Colored areas where used to calculate the mean molecular weight and standard deviation. (F) SEC-MALS of TAp63αmin in 2 M urea (preincubated in 2 M urea for 14 min at RT). (G) SEC-MALS of TAp63αmin in 2.5 M urea (preincubated in 2.5 M urea for 25 min at RT). (H) WB and corresponding bar diagram of pull-down experiments with ΔNp63α, TAp63α R604E R608E and TAp63α incubated either during or after expression in RRL at 30°C for 1.5 hr with His6-tagged p73 TD or a mutant that is not able to form hetero-tetramers (His6-p73 TDHOMO). Pull-down is achieved by hetero-tetramerization of His6-tagged p73 TD with specified p63α constructs. Quotient of pull-down (P) and input (I) is shown relative to TAp63α incubated after expression with p73 TD (set to 1). Pulldowns were performed in technical triplicates and error bars denote standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.13909.017

Figure 4.

Figure 4—figure supplement 1. Urea treatment of p63 structured domains.

Figure 4—figure supplement 1.

To prove that moderate concentrations of urea (up to 3 M) do not unfold domains inside TAp63αmin, individual domains were incubated for 1 hr at different urea concentrations and subjected to size exclusion chromatography (SEC) on a Superdex 75 3.2/300 column at corresponding urea concentrations. SEC profiles of TD (A), SAM (B) and DBD (C) at urea concentrations of 0 M, 1.75 M and 3 M urea.
DOI: http://dx.doi.org/10.7554/eLife.13909.018
Figure 4—figure supplement 2. Urea unfolding experiments with TAp63αmin.

Figure 4—figure supplement 2.

(A,B) TAp63αmin samples were incubated for 1 hr at different urea concentrations and subjected to size exclusion chromatography (SEC) on a Superose 6 3.2/300 column at corresponding urea concentrations. As in Figure 4A but at higher urea concentrations. At a urea concentration of 4 M urea the tetramers seem to unfold as seen in the partial shift to higher elution volumes. (C) TAp63αmin was incubated in 1.75 M urea for 24 hr and injected onto a Superose 6 3.2/300 column equilibrated in 1.75 M urea.
DOI: http://dx.doi.org/10.7554/eLife.13909.019