Modulating Protein-Nanoparticle Interaction Energetics using Site Directed Mutagenesis

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Nanoparticle technology has been a growing field in biomedical research. This is in part due to potential applications in drug delivery, biosensing, diagnostics, and imaging. Our long-term goal is to use protein functionalized AuNPs as a general tool for molecular sensing and drug delivery. The ability to use nanomaterials as biosensors and drug delivery methods in cellular uptake is directly dependent on the amount of protein that is able to bind to the surface of any given nanoparticle. It is hypothesized that electrostatic interactions play a significant role in protein-AuNP interactions, since citrate-stabilized AuNPs carry a net negative charge. Our group has developed an NMR-based approach to rapidly quantify bound protein to AuNP. To understand the above phenomenon, GB3 was chosen as our model protein and it contains seven lysine residues. These positively-charged lysine residues are involved in protein-AuNP binding, and a potential binding site was identified using APBS calculations which contain lysine residues. This hypothesis was tested by mutating the lysine residues to alanine one at a time using site-directed mutagenesis. NMR experiments were carried out to observe how the binding capacities of each of these variants change relative to wild type GB3. Notably K4A, K13A and K50A variants has significantly reduced binding, while the binding capacity of other lysine to alanine variants was on par with wild-type GB3. To obtain better understanding, GB3 variants were competed with wild type GB3 in the same solution with AuNP to observe how the binding capacities vary with wild type GB3. As predicted, the binding capacity ratio was lower for lysine residues in the proposed binding site were changed to alanine. A reduced binding capacity ratio was not observed for other lysine variants. The results reported are significant in establishing our original hypothesis, and suggest that GB3 adopts a specific orientation on the AuNP surface.