Thermodynamic stability of wild-type and mutant p53 core domain

Abstract

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn2+ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25 degrees C and 9.8 kcal/mol at 10 degrees C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.

Figure 1

DNA binding analysis of p53 core domain from DSC. Thermograms were determined in 50 mM Tris, pH 7.2/1 mM DTT of (top trace) double-stranded DNA (dsDNA) oligonucleotide (7 μM) (T_m = 56°C), (middle trace) p53 core domain + dsDNA oligonucleotide equimolar (T_m = 49°C), (bottom trace) p53 core domain (7 μM) (T_m = 42°C) alone. Data are shown without correction for the buffer baseline and are offset for clarity.

Figure 2

Fluorescence of p53 core domain in 50 mM sodium phosphate, pH 7.2/5 mM DTT at 25°C on excitation at 280 nm. The native state (N) has a tyrosine emission maximum at 305 nm, which shifts to 310 nm on denaturation. There is a clear tryptophan emission maximum for the denatured state (D) at 350 nm. The tryptophan fluorescence of N is very weak. D and N have the same fluorescence at 321 nm. Aggregated states have a fluorescence maximum at 340 nm; shown is the emission of p53 core domain after being heated to 60°C and cooled to 25°C.

Figure 3

Scheme for the equilibria between the native and denatured states of p53 core domain with and without bound zinc. The equilibrium that has been measured is shown by K_D-N^H.

Figure 4

Urea denaturation of wild-type (WT) and mutant p53 core domain monitored by normalized fluorescence emission at 356 nm (data without normalization are shown for R175H; excellent data are obtained in general without normalization). Data are plotted as fraction unfolded and fitted to Eq. 1. Protein was buffered in 50 mM sodium phosphate, pH 7.2/5 mM DTT. An excitation wavelength of 280 nm was used.

Figure 5

Molscript (22) picture of p53 core domain. Residues mutated are labeled and shown as stick models as is Trp-146, which was used as a fluorescent probe for denaturation.