The inherent electric field imposed by a protein scaffold allows for the ability to improve the efficiency of a reaction. In this project, a joint collaboration with an experimental group at Columbia University, we aim to use site-directed mutagenesis on a functionalized biotinylated rhenium phenanthroline complex and tetrameric streptavidin scaffold to enhance and tune its catalytic efficiency to aid in the photoreduction of CO2 to CO. Through molecular dynamics simulations, density functional theory calculations on small cluster models, and hybrid quantum mechanical/molecular mechanics calculations on the full protein, we show a relationship between access to the active site and experimentally determined CO2 turnover. Additionally, our data suggests that the presence of a positively charged cation, or, to a lesser extent, the presence of a hydroxyl group, near the active site is important to improving the stability of CO2 binding, These results demonstrate the improvement in photoreduction in the streptavidin complex over the free phenanthroline in solution, further illustrating a protein scaffold’s ability to improve catalysis. Through this analysis, we have been able to predict other promising mutants, such as the S112K variant.

Enzymes catalyze a remarkable diversity of chemical transformations, leading to the recent emergence of biocatalysis as an alternative to traditional chemical synthesis. Biocatalysis offers several advantages over traditional synthetic methods: milder reagents, better atom economy, improved stereoselectivity, and minimized hazardous waste products. Importantly, enzymes catalyze reactions not possible or impractical through current synthetic means. Enzymatic traits such as activity and selectivity can be improved through directed evolution using iterative mutagenesis to further specialize catalysis. In this study, we evolve a metalloenzyme to catalyze an enantioselective radical reaction. After three rounds of mutagenesis leading to three active site mutations, we generated a variant giving 11% product yield (from 1%) and an 83:17 enantiomeric ratio, with a total turnover number of 95. These findings continue in demonstrating the potential of biocatalysis to enable previously inaccessible transformations and the utility of directed evolution in expanding enzyme functionality.

Affinity purification mass spectrometry (AP-MS) is a technique that allows for the detection and identification of protein-protein interactions (PPIs) present for a specific protein. PPIs provide insight into the pathways that a protein is involved in, which can be used to determine a proteins’ biological function. Previous work in the Backus Lab established a proteomic platform called neo-N-terminal SP3 pyridine carboxaldehyde enrichment (N-SPyCE), which enriches and labels protein neo-N-termini. This method revealed that during T cell stimulation, polypyrimidine tract binding protein 1 (PTBP1) is cleaved into various proteoforms by the protease Legumain. PTBP1 contains various RNA recognition motif (RRM) domains that classify it as an RNA binding protein (RBP). The proteoforms of interest for my project are a full length PTBP1, PTBP1 cleaved at S35, and PTBP1 cleaved at V304. I am creating recombinant constructs of each proteoform with either an N-terminus flag tag or a C-terminus flag tag. I will perform a flag pulldown with each of these constructs transiently expressed in HEK cells to enrich for the PTBP1 proteoform and its direct interactors, and identify these interactors via mass spectrometry. As PTBP1 is a known RBP, I hypothesize that PTBP1 and its proteoforms interact with other RBPs and splicing factors that are involved in determining T cell state. The interactors identified will aid in determining the biological role of each proteoform.

Macrophages play an important role in the innate immune response, typically adjusting the relative amounts of inflammatory and reparative processes. Because of their high mobility and frequent interactions with the embedding tissue, accurate, non-invasive phenotyping of intact macrophages is essential in studies of their functional roles in various contexts and to help advance targeted therapeutic strategies. Current phenotyping methodologies are primarily invasive or entail terminal endpoints, preventing the analysis of dynamic phenotypic transitions in live cells. Herein, we introduce a robust, non-invasive approach for phenotyping live macrophage cells leveraging high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and a simple classification algorithm. Our method can unambiguously classify M0, M1, and M2 macrophage phenotypes using data derived from distinct peaks in the 1H NMR spectrum. Using the method of dictionary learning, we compressed the dimensionality of the embedding space to two, facilitating subsequent vector clustering. The identified phenotypes exhibited non-overlapping distributions at 12 hours and 48 hours post-activation, suggesting the potential for temporal resolution. This constitutes a new method for phenotyping live cells, which does not require fixing cells or chemical treatment. The real-time spatiotemporal monitoring of phenotype could potentially help understand immunological and inflammatory processes, as well as response to treatment and other stimuli such as environment.

Proteins naturally accumulate covalent damages to their structure over time, eventually causing proteostasis decline within a cell. One example are L-isoaspartyl damages formed by the spontaneous isomerization of aspartate residues. Repair mechanisms exist within a cell to combat the accrual of these L-isoaspartyl damages, such as the PCMT1 enzyme which uses AdoMet to methylate and repair L-isoaspartate. However, recent studies suggest that an alternative mechanism may exist. PCMTD1 is predicted to act as an E3 ubiquitin ligase to aid in the proteasomal degradation of L-isoaspartyl damaged proteins within a cell. It contains binding motifs for L-isoaspartate and AdoMet, similar to PCMT1, but also for ubiquitin ligase recruitment. The Clarke group is currently investigating this protein and its role in proteostasis through structural and functional characterization methods. In this study, we use tetrazine click chemistry reactions to conjugate GFP to L-isoaspartyl peptides, and then investigate binding capabilities of PCMTD1 to these proteins via native top-down mass spectroscopy (nTDMS). We find that PCMTD1 preferentially binds to proteins containing L-isoaspartyl residues, validating its role as an E3 ubiquitin ligase specific to L-isoaspartyl damaged proteins.