Biophysical characterisation and rescue of p53 cancer mutants

The aim of this thesis was to use biophysical methods to characterise the stabilities and DNA binding properties of monomeric and tetrameric p53 cancer mutants, and to study various approaches aimed at rescuing structural mutants of p53. A detailed study of the destabilising effects of p53 mutations was performed using differential scanning calorimetry and urea denaturation, while equilibrium binding of p53 mutants to a specific promoter sequence, gadd45, was studied using fluorescence anisotropy and analytical ultracentrifugation. This thesis will also discuss how p53 structural mutants may be rescued by suppressor mutations that either increase the overall protein stability of compensate specifically for oncogenically induced loss of interactions. Stability and DNA-binding measurements showed that the destabilising effects of mutations H168R and R249S were not additive, and that these mutations in combination restored DNA binding. Similar biophysical techniques were used in analysing a series of p53 core domain mutants in which the residue Ser-116 in the middle of flexible loop L1 was mutated. One mutant, S1116C, was found to be more stable than previously predicted. The crystal structure showed how the mutation had led to formation of a new hydrogen-bonding network. Altogether, these protein-engineering studies provided useful insights into possible ways to rescue p53 function. A small set of compounds was selected based on the nature of proteins and peptides that were known to interact with p53. NMR spectroscopy was used to screen for compounds binding to the protein target and to probe for atomic detail of binding interactions. It was shown that 15N-1H HSQC could be used for screening and deconvoluting mixtures of compounds, in the presence of 5% v/v d6-DMSO, at relatively high throughput.EThOS - Electronic Theses Online ServiceGBUnited Kingdo