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Modeling Mechanical Processes of DNA-Interacting Protein Complexes Synthesizing Studies of the Binding and Catalytic Behavior of Constituent Subunits
Morgan, Aaron
Morgan, Aaron
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Abstract
I began my graduate research with a study of Chromatin remodelers, which are molecularmotors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. Here I present a quantitative analysis of nucleosome repositioning by the chromatin remodeler ISWI and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assaydependent limitations can bias studies of nucleosome repositioning. Together, these results suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself. I moved from this seemingly inefficient system to a remarkably efficient system, the B. subtilis Φ29 bacteriophage. The gp16 ATPase is the constituent subunit of the pentameric dsDNA (Double-stranded Deoxyribonucleic Acid) translocation motor of the B. subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of (523.8 ± 247.3) nM for ATP and a lower limit of 30μM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.
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Date
2022-12-31
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University of Kansas
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Keywords
Biophysics, Bacteriophages, Chromatin Remodelers, DNA, Fluorescence Spectroscopy, Molecular Motors, Protein Interactions