THERMODYNAMIC STABILITY OF WILD-TYPE AND MUTANT P53 CORE DOMAIN

Some 50% of human cancers are associated with mutations in the core do main of the tumor suppressor p53, Many mutations are thought just to d estabilize the protein, To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain an d its mutants, The use of differential scanning calorimetry or spectro scopy to measure its melting temperature leads to irreversible denatur ation and aggregation and so is useful as only a qualitative guide to stability, There are excellent two-state denaturation curves on the ad dition of urea that may be analyzed quantitatively, One Zn2+ ion remai ns tightly bound in the hole-form of p53 throughout the denaturation c urve, 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 condi tions, the wildtype domain forms an aggregate that is relatively highl y fluorescent at 340 nm on excitation at 280 nm, The destabilized muta nts give this fluorescence under milder denaturation conditions.