The tumor suppressor protein p53 is a master regulator of cell fate. The activity of p53 is controlled by a plethora of posttranslational modifications (PTMs). However, despite extensive research, the mechanisms of this regulation are still poorly understood due to a paucity of biochemical studies with p53 carrying defined PTMs. Here, we report a protein semi-synthesis approach to access site-specifically modified p53. We synthesized a set of chemically homogeneous full-length p53 carrying one (Ser20ph and Ser15ph) or two (Ser15,20ph) naturally occurring, damage-associated phosphoryl marks. Refolding and biochemical characterization of semisynthetic p53 variants confirmed their structural and functional integrity. Furthermore, we show that phosphorylation within the N-terminal domain directly enhances p300-dependent acetylation approximately twofold, consistent with the role of these marks in p53 activation. Given that the p53 N-terminus is a hotspot for PTMs, we believe that our approach will contribute greatly to a mechanistic understanding of how p53 is controlled by PTMs.
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