Graduate Research - Bioconjugation Reaction Development

The synthetic modification of proteins plays an important role in the fields of chemical biology and biomaterials science. As applications of protein-based materials continue to become more complex, improved methods for the covalent modification of proteins are needed. Although many methods for the modification of native and artificial amino acids exist, they often require long reaction times or lengthy syntheses of reactive substrates. To address this need I initiated the development and application of a suite of bioconjugation reactions that utilize ortho-aminophenols. The oxidative coupling of aniline residues with o-aminophenol substrates was optimized. Potassium ferricyanide was identified as an alternative, mild oxidant for this coupling. These new conditions enabled the use of the oxidative coupling reaction in the presence of free cysteines and glycoslated substrates. Aminophenols were also discovered to react with native residues on protein substrates in addition to artificial aniline moieties under certain conditions. Cysteine and the N-terminus were identified as the reactive residues. The oxidative coupling of o-aminophenols with the N-terminus was optimized to achieve high levels of modification on peptide and protein substrates. The oxidative coupling of anilines and o-aminophenols was applied to the synthesis of a targeted, virus-like particle and to the detection of protein tyrosine-nitration. Overall, these updated and novel oxidative coupling methods expand the utility ortho-aminophenols for the modification of proteins.

Graduate Research - Targeted Imaging

Cardiovascular diseases present a serious threat to human health, with nearly one in three deaths in the United States attributable to these afflictions. The development of atherosclerosis targeted imaging agents has the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of cardiovascular diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. I synthesized chemically functionalized bacteriophase MS2 capsids as protein-based nanoparticles for fibrin and vascular cellular adhesion molecule 1 (VCAM1) imaging. The capsids were modified using an oxidative coupling reaction, conjugating ~90 copies of targeting peptides to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents.

Postdoctoral Research - Protein-Polymer Biocatalysts 

Proteins comprise one of the most impressive categories of polymers known: they produce extremely strong and tough materials, they efficiently catalyze chemical transformations, they selectively bind analytes within complex mixtures, and they harvest light by converting it into chemical energy. By combining the incredible diversity of structure and function of proteins with the stability, chemical diversity, and processability of synthetic polymers, materials with the advantages of both components can be accessed. In order to use proteins as sensors, biofuel cells, or industrial catalysts the protein-based material must maintain mechanical integrity, protein stability and longevity, and access to the protein active site. The self-assembly of proteins in solid-state materials has the ability to achieve each of these requirements. 

Two methods for the self-assembly of protein biocatalysts were explored, both relying on the ability of block copolymers to direct self-assembly. First, synthesis of a protein-polymer block copolymer was achieved by site-specific functionalization of cytochrome P450 BM3 with poly(N-isopropylacrylamide). The polymer modified protein generates a material that can be easily processed to create a self-assembled heterogeneous biocatalyst. The self-assembly of the bioconjugate in concentrated solutions was characterized by small-angle x-ray scattering (SAXS) and depolarized light scattering (DPLS). The thin film nanostructure was also investigated by grazing-incidence small-angle x-ray scattering (GISAXS). Catalytic activity of the thin films for C-H oxidation was measured using a variety of substrates.

An additional method for controlling the self-assembly of proteins was probed. Non-specific modification of proteins to vary their surface charge was also examined as a means for directing protein self-assembly. Using model proteins and lysine succinylation, a panel of proteins with varying charge density was synthesized. The ability of these proteins to form complex coacervates with oppositely charged polyelectrolytes was studied in order to identify parameters that governed complex coacervation of proteins. The fundamental parameters uncovered allow any protein of interest to be successfully modified to enable complex coacervation. These findings were applied to the synthesis of complex coacervate core micelles with protein cores. Encapsulation of P450 enzymes in these ionic micelles enables their use as biocatalysts for C-H oxidation.