QUANTIFYING THE ENERGETICS OF BINDING BETWEEN p53 AND DNA USING ELECTROPHORETIC MOBILITY SHIFT ASSAYS
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A new class of proteins has been termed “intrinsically disordered” or “ID” for short. The proteins within this class appear to lack tertiary stability, though protein structure has been linked to function in the past. These ID proteins are also prevalent, especially in eukaryotic proteins (~30%) and transcriptional factors (~70%). The high occurrence of ID suggest that this natively unfolded motif has been evolutionary selected. One of these ID proteins is the tumor suppressor protein p53. The p53 protein is a transcription factor that plays a role in ensuring genomic integrity. p53 is a homotetramer made up of seven domains with only two of the seven domains being natively folded. The other five domains lack tertiary structure under physiological conditions. It has been seen that these ID domains play a role in p53 regulation, suggesting allosteric communication. p53 is highly studied due to its connection with cancer. In approximately 50% of human tumor cells there is a mutation within the p53 gene, leading to p53 to be highly studied. Because of p53’s connection with cancer a database showing the frequency of mutations that effect p53 function is already available, making p53 a plausible model for how the ID motif plays a role in regulatory mechanisms. To under stand how p53 transduces signals a mutational screen on binding energetics will be done. Before this bigger project and to show practicality of its success, basic protocols need to be established. The main focus of this thesis is exactly that. In this body of work its shown that wild type p53 and hyperstable p53 can be expressed, purified and used in binding studies. While the wild type p53 seems to be unpredictble due to its ability to form tetramers, the hyperstable p53 shows promising results for continuation with the mutational screen.