Mapping the Solvent Accessible Surface Area of Proteins using Pyrite Nanocrystals: Bringing Oxidative Footprinting onto the Bench
Leser, Micheal Jefferson
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Mapping the Solvent Accessible Surface Area of Proteins using Pyrite Nanocrystals: Bringing Oxidative Footprinting onto the Bench Mapping a protein's solvent accessible surface provides insight into structure and assembly reactions when atomic resolution information is not available. `Oxidative footprinting' is an indirect mapping method that measures a residue's solvent accessibility via hydroxyl radical side chain oxidation. The studies presented hi this thesis describe the fabrication, validation, and application of a novel approach to the generation of hydroxyl radical in solution. A pyrite nanocrystalline surface deposited on commodity shrink-wrap film provides an accessible, cost effective alternative to radiolysis, photolysis, and electrochemistry for generation of hydroxyl radical. Simple chemical generation of the hydroxyl radical is achieved by means of the Fenton reaction by incubating hydrogen peroxide on the surface of Pyrite Shrink-Wrap Laminate. The thermolabile nature of the plastic base material allows sample wells to be molded in standard microtiter plate format for use with a standard laboratory pipette during depositing and removal of sample volumes as small as several microL. Multiplexing protein oxidation requires only a multichannel pipette. Measurement of the degradation of a fluorescent dye demonstrates quantitative dose-response oxidation curves is generated with Pyrite Shrink-Wrap Laminate as a function of either reaction time or hydrogen peroxide concentration. This novel method of oxidative footprinting was applied to the study of the immunological regulatory protein Programmed Death 1 (PD-1) and its interaction with a natural ligand, PD-L1. The interaction between PD-1 and PD-L1 is an inhibitory signal to curb the activation of T-cells after an initial immune response to invading pathogens. These proteins are important therapeutic targets for cancer and autoimmune disorders. Our work demonstrates the potential of Pyrite Shrink-Wrap Laminate as a low-cost method of oxidative footprinting scalable to high throughput implementation.