Abstract

The tumour suppressor protein p53 orchestrates the response to cell damage and thus plays a central role in preventing cancer. p53 is tightly regulated by post-translational modifications (PTMs). The precise mechanisms through which p53 PTMs operate are difficult to elucidate due to the complexity of p53 signalling and challenges associated with preparing site-specifically modified p53 for in vitro studies. To address these issues, we have developed a protein semi-synthesis strategy to access p53 in defined PTM states. Using such ‘designer’ phospho-p53 variants we have probed the mechanism of p53 activation through phosphorylation in vitro. Moreover, we found that a spontaneous protein backbone modification, the rearrangement of an asparagine to an isoaspartate residue, reconfigures p53’s binding partner specificity, which suggests that p53 could act as a molecular time bomb. Given the importance of PTMs in p53 signalling, we believe that our chemistry-driven approach will contribute greatly to a mechanistic understanding of how this protein makes cellular life and death decisions.