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Evaluation of Energetics-based Techniques for Proteome-Wide Studies of Protein-Ligand Binding Interactions

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Friday, March 27, 2015
12:30 pm - 3:30 pm
M. Ariel Geer, Ph.D. candidate
Ph.D. dissertation defense

A series of energetics-based techniques for the assessment of protein stability and ligand binding have been developed over the past 10 years. While the capabilities of the energetics-based techniques have been exhibited using a few model systems, the scope of the techniques to a wider range of biological questions remained to be shown. The Stability of Proteins from Rates of Oxidation (SPROX) technique and the Pulse Proteolysis (PP) technique were successfully applied to the discovery of known and novel protein-protein, protein-ATP, and protein-drug interactions in yeast and human cell lysates. The false-positive rate of SPROX protein target discovery was determined to be < 0.8% for analyses performed on orbitrap mass spectrometer systems. The SPROX and PP techniques were used to evaluate the interactions of geldanamycin and gedunin, two Hsp90 inhibitors with different modes of action. Manassantin A interactions were assessed in hypoxic MDA-MB-231 cell lysates using SPROX and PP, and the previously unknown protein targets Filamin A and B were detected as hit proteins using both experimental protocols with different quantitation strategies. In this work, the range of systems to which the energetics-based approaches have been applied has been expanded to evaluate the scope and reproducibility of the techniques and to discover novel protein-ligand interactions.