Ribonucleotide reductase subunit 2-like ligand binding oxidase (R2lox) is a Mn/Fe enzyme that is highly upregulated in Mycobacterium tuberculosis. Its physiological function is unknown, but R2lox performs a 2-electron oxidation with its own protein scaffold as a post-translational modification, where an inert C-H bond is cleaved. When heterologously overexpressed in Escherichia coli, R2lox co-purifies with a fatty acid bound in its hydrophobic channel, coordinating to the Mn/Fe center. It’s unclear if the fatty acid is a purification artifact or functionally relevant. To understand this, the A171F R2lox mutant is used, mutating an alanine to a phenylalanine which prevents fatty acid copurification, rendering the channel accessible. In its reduced MnIIFeII state, R2lox reacts with O₂ to form a Mn(IV)Fe(IV) intermediate that drives a two-electron oxidation, producing a Tyr-Val ether crosslink near the active site to complete its first turnover. Proteomics results quantifying the cross-link formed in A171F versus wildtype will be discussed, informing on the role of the fatty acid in the catalytic mechanism of R2lox. Gas chromatography-mass spectrometry assays were performed to evaluate R2lox activity with potential substrates, including aldehydes, under turnover conditions. A comparison of A171F to wild type R2lox shows diminished crosslink formation and a lack of reactivity toward long-chain aldehydes and suggests that the fatty acid facilitates ether crosslink formation and plays a functional role in the catalytic mechanism.

Wnt/ß-catenin signaling is critical for cell growth and development, with its hyperactive dysregulation implicated in the development of cancer. Current therapeutic research on inhibition of Wnt/ß-catenin signaling is impeded by the high cost of experimentally determining binding affinities. Consequently, interest has risen in screening potential inhibitors binding affinities with computational tools to reduce costs. Here, we test the validity of a computational molecular dynamics simulator, Binding Free Energy Estimator 2 (BFEE2), and a parameterization software, CGenFF, in determining peptide and small molecule ligand affinity for Wnt/ß-catenin signaling. We focus on the Dishevelled (DVL) PDZ domain, a key mediator in WNT signaling through its ability to bind to various peptide ligands. We analyze the binding affinities of several DVL PDZ domain-peptide and domain-ligand complexes against previously established results to determine the validity of computational analysis. We conclude that computational molecular dynamics simulations are moderately effective in determining DVL peptide ligand complex affinity with an R2 of 0.44; however, the simulation was not effective with small molecule drug inhibitors with an R2 of 0.06.

Cryogenic electron microscopy (cryoEM) has emerged as a critical technology for imaging biomolecules. Despite the maturity of the technique and its broad application across basic and applied biosciences, it remains challenged by technical limitations of the instrumentation. One such limitation—coincidence loss (CL)—arises when multiple incident electrons arrive at a detector within a single readout frame and are recorded as a single event. This produces a nonlinear relationship between the true electron dose rate and the measured count rate, leading to systematic undercounting and reduced image resolution. I therefore chose to investigate whether post-acquisition computational methods can mitigate CL and recover information lost during detection. I developed correction algorithms that model detector dose-response behavior and statistically estimate missed electron events. These methods were applied to publicly available datasets from EMPIAR (EMPIAR-10020 and EMPIAR-11261). Corrected datasets were first evaluated in Python to verify global count statistics and pixel intensities, then processed through reconstruction pipelines in CryoSPARC and compared with uncorrected data. Preliminary results suggest that CL correction is possible in moderate-to-high CL regimes and that effective approaches must account for local pixel statistics as well as global dose averages. Reliable CL correction could allow cryoEM experiments to operate at higher dose rates while preserving structural information, improving data collection efficiency.

Tight junctions are adhesion complexes formed between endothelial and epithelial cells whose formation depends on scaffolding protein ZO (zonula occludens)-1. ZO-1 recruits certain transmembrane proteins to tight junctions via interactions with the C-terminal domains (CTD) and has been experimentally shown to bind to occludin (OCLN) and other tight junction proteins using its SH3 and GUK domains via co-immunoprecipitation, as shown in Fanning et al. (1998), but this interaction has yet to be structurally characterized. As such, our goal is to use structural characterization to elucidate the competitive interactions of ZO-1 and other TJ proteins to understand TJ assembly. To start, OCLN CTD and ZO-1 will be shown to interact using a size exclusion column. After confirming binding, next steps will include adding on the C-terminal tails of TAMPs and other auxiliary cytosolic proteins known to interact with ZO-1 to build a complex. By creating as large of a ZO-1 complex as possible, we hope to understand how tight junction assembly occurs mechanistically. A stronger understanding of tight junction formation can help us identify therapies for various diseases caused by tight junction dysfunction, including inflammatory bowel disease, hepatitis C, and certain cancers.