Browsing by Subject "Protein Corona"

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    Molecular level investigation of the spatial and temporal nanoparticle-corona complex through the application of techniques used to investigate protein-protein interactions
    (2024-09) Northwick, Andrew
    Engineered nanoparticles have been part many advances in electronics, agriculture, and medicine off the back of the unique properties from their size. On this scale, these materials have direct interactions with proteins, cells, and other biomolecules and adsorb a coating called a corona. The corona modifies the nanoparticle surface and functionality and has been shown to impact colloidal stability and function. Research had identified that the surface charge of the nanoparticle affects the rate of corona adsorption and that the concentration of a protein in solution does not correlate to the concentration in the corona. However, many of these studies are hampered by the fact the corona adsorbs non-covalently and that techniques required to isolate the complex for analysis remove all but the strongest interactions. This is not a novel challenge; techniques have been developed to protein-protein interactions (PPI) at the molecular scale that interact through similar mechanisms. In this dissertation, we outline the adaptation of two techniques, protein footprinting and photoactivated crosslinking, to investigate two aspects of the corona that have gone uninvestigated, the spatial and temporal corona. We have previously shown that this can be achieved though lysine footprinting, looking for decreases in labeling representing a loss of solvent accessibility through the adsorption to the nanoparticle. Using cytochrome C as a model, we found two binding sites and that other sections of the protein deform and become more solvent accessible. We expand upon this work investigating how protein deformability and matrix effect change binding. We find that proteins prefer to adsorb to the surface of the nanoparticle through binding loop regions and that the observed protein deformation is likely the rotation of secondary structure to accommodate optimal binding. (Chapter 2). With a baseline of protein binding sights, we then investigate how protein-protein interactions alter the corona orientation to start applying this protocol to more biologically relevant conditions (Chapter 3). We find that the presence of multiple proteins does not change the binding sites but does impact the packing on the surface of the nanoparticles. Addressing how the corona changes as it develops the hard corona presents a more difficult challenge as any initial contacts are not strong enough to survive isolation from solution, much like PPI. To isolate these interactions, photoreactive crosslinkers have been developed to form a covalent bond between targets. We outline a design synthetic method to incorporate a photoreactive group into a ligand to incorporate onto the nanoparticle surface (Chapter 4). We also investigated a crosslinker with reported <1 min activation times to capture interactions as early as possible and found that the presence of nanoparticles provides interference. We also highlight the potential colorimetric assays to measure and optimize the activity of the photocrosslinkers pushing forward to investigate the corona formed from complex mixtures.