Regulation of transcriptional activation by protein disorder

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Title: Acquisition of a fluorescent plate reader for measuring protein-protein and protein- DNA binding affinities Summary Understanding protein-protein and protein-DNA binding is required for a deep understanding of protein function and its relation to disease. Fluorescence polarization (FP) is an outstanding tool for measuring protein-protein and protein DNA binding affinities, and it is more flexible than isothermal titration calorimetry (ITC). Modern FP instruments can measure binding affinities whether heat is produced and use a microplate reader for high throughput and rapid detection. We request funds for the Agilent BioTek Synergy H1 microplate reader to improve our ability to measure binding affinity between p53 mutants and DNA over a range of salt concentrations where a heat signal from ITC cannot be detected. Results from these experiments will improve our understanding of how p53 DNA binding specificity is regulated by an intramolecular interaction between the transactivation and DNA binding domains, which is one of the main goals of the parent award (R01GM115556).