Phosphoserine Incorporation into the Intrinsically Disordered N-terminal Domain of the p53 Tumor Suppressor Protein
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Protein phosphorylation is used biologically as a mechanism to control many critical life processes. Since phosphorylation usually occurs within protein regions that are intrinsically disordered, quantitative and structural descriptions of phosphorylation effects in intrinsically disordered proteins (IDPs) are needed to understand the molecular basis of key aspects of development, aging, and disease. A few studies have investigated the structural effects of phosphorylation in IDPs but, overall, this subject remains mostly uncharacterized. The goal of this thesis project is to adapt literature-reported recombinant techniques for obtaining phosphorylated protein for use in our structural studies of the intrinsically disordered N-terminal region of the p53 tumor suppressor protein, p53(1-93). Healthy p53 activity is regulated by phosphorylation at sites in its disordered N-terminal region, whereas aberrant p53 activity has been linked to numerous human cancers. To obtain phosphorylated p53(1-93), two strategies were employed. The first used a commercially available kinase known as DNA-dependent protein kinase, that is thought to phosphorylate positions 15 and 37. The second is an orthogonal translation system consisting of two independent plasmids and genetically recoded Escherichia coli (E. coli). One plasmid is used for expression of p53(1-93) and the other for a phosphoserine (Sep) tRNA-synthetase system to permit phosphoserine incorporation into p53(1-93). Sep tRNA utilizes the anti-codon corresponding to TAG and, accordingly, recognizes the TAG stop codon. Two E. coli strains that were used have genes for serine phosphatase deleted. One strain has had all TAG stop codons recoded as TAA and release factors corresponding to TAG silenced. Genes for expressing RNA synthetase and Elongation Factor specific to Sep tRNA are included in the Sep tRNA-synthetase plasmid. The TAG codon will be substituted into the p53(1-93) gene at positions corresponding to serine 15, 33, and 46 to mimic phosphorylation at those sites.