Chemistry Honors Theses

NAD(P)H:quinone oxidoreductases (NQOs) are flavin-dependent enzymes that catalyze the two-electron reduction of quinones and the oxidation of NAD(P)H. NQOs play a role in cellular detoxification by preventing the formation of radical quinones thus avoiding the generation of reactive oxygen species. The NQO from Pseudomonas aeruginosa PAO1 (PA1024) is proposed to serve a dual function in the cell by detoxifying quinones and balancing the [NAD⁺]/[NADH] ratio. Crystal structures of wild-type PA1024 were previously solved in free form and in complex with NAD⁺ (PDB: 2GJN & 6E2A). Comparison of the ligand-free and ligand-bound structures of the enzyme reveals a loop (residues 75-86) in two conformations, suggesting the mobile loop may play a role in NADH binding and selectivity. P78 is located on the mobile loop and is hypothesized to provide internal rigidity to the mobile loop to help carry out its functions. In the present study, site-directed mutagenesis was utilized to replace P78 with glycine and increase mobile loop flexibility. The mutant (NQO-P78G) was expressed and purified as previously described for wild-type PA1024. Apparent steady-state kinetics of NQO-P78G, at varying concentrations of benzoquinone and a fixed [NADH] of 0.1 mM, showed a 20-fold reduction in the k_cat value with respect to the wild-type enzyme, i.e., 1.3 s^-1 vs. 27 s^-1. When the concentration of NADH was varied with a fixed [benzoquinone] at 21 μM, NQO-P78G could not be saturated and only a k_cat/K_NADH value of 11,000 ± 700 M^-1 s^-1 could be determined. On the basis of kinetic results presented here, we propose that P78 does not play a role in substrate specificity but rather it is important in assisting with catalysis in PA1024.

Ribosomes are a key component of the protein homeostasis (proteostasis) network that controls protein synthesis, folding, and degradation, driving functional diversity and nervous system complexity. Proteostasis maintenance is challenging in neurons due to their unique morphology, which directly impacts neural connectivity and signal propagation. The importance of neuronal architecture in regulating nervous system function is highlighted by proteinopathic diseases and other neurological disorders that stem from dendritogenesis dysfunction.

Historically, ribosomes were thought to be homogenous machinery, but recent studies have instead suggested the concept of “specialized ribosomes,” which posits that ribosomes preferentially translate select mRNAs based on several factors, such as ribosome composition in different cell types. However, the mechanistic role of ribosome heterogeneity in regulating dendritic morphology remains unknown. Dissecting the impact of specialized ribosomes on morphology and neuronal function could be key to understanding disorders linked to ribosomal dysfunction, such as autism and microcephaly.

To address this, our lab previously conducted cell-type-specific morphological screening of thirty ribosomal proteins (RPs) in CI and CIV neurons in Drosophila melanogaster. Individual RPs were knocked down using the GAL4/UAS binary system, and the results showed that, while most knockdown affected dendritic morphology in both classes, select RP knockdown had a cell-type-specific effect. The effect of RP knockdown on ribosome localization along the dendritic arbor and at branch points was also investigated. Our results suggest that knockdown of RPs that disrupt dendritic morphology also causes defects in ribosome trafficking, indicating a cell-type specific requirement of RPs in maintaining neuronal architecture.

To determine the putative effects of ribosome heterogeneity on global protein translation, fluorescent non-canonical amino acid tagging (FUNCAT) was performed. When combined with cell-type-specific gene expression, FUNCAT allows for class-specific protein visualization and quantitative analysis of protein translation. Additionally, a preliminary ribosomal protein conservation study was performed to determine if certain ribosomal proteins are functionally conserved across species.

Chemokine receptor type 4 (CXCR4) is overexpressed in cells associated with various disease pathways such as autoimmune disorders, inflammation, HIV-1 proliferation, and cancer. Healthy functions such as stem cell differentiation and migration are linked with the CXCR4/CXCL12 signaling pathway. Hence, the binding interaction of CXCR4 with its natural cognate CXCL12 ligand can be partially blocked with small molecules to hinder the mechanism of cancer metastasis or inflammation but still allow normal cell functions to occur. Various antagonists and modulators for CXCR4 have been synthesized and tested, but new compounds with better efficacy are necessary due to cardiotoxicity and/or poor oral bioavailability of these compounds. The overall goal for this research is to synthesize asymmetrical, pyridine-based and thiophene-based compounds as potential modulators of CXCR4-CXCL12 activity. A new research scheme is being investigated and tested using 2,5-thiophene as the central ring and modifications to the side chains with a different hit functional group on each side to yield an asymmetrical small molecule antagonist. After the synthesis process is completed, the analogues are analyzed through NMR and MS and proceed to be tested with further biological tests.

This study examined the propensity for a weathered muscovite-rich test material to sorb 137Cs in a dilute NaCl solution (1 mmol/L, pH 5) across a range of added stable Cs and Rb concentrations for 130 days at room temperature. This muscovite test material, slaked from processed kaolin ore, was composed of 76% muscovite, 21% kaolinite and 3% quartz. Sorption experiments in the absence of stable Cs and Rb yielded increasing Kd values (1.49 x 103 mL/g to 1.18 x 104 mL/g) over 130 days for 137Cs sorbed onto muscovite. Sorption experiments with stable Cs and Rb displayed linear decreases in Kd values as functions of the concentrations of stable Cs and Rb. These findings are consistent with a Freundlich isotherm. After 130 days, the addition of NaCl (1 mM and 10 mM) caused the desorption of only a small fraction (0.011% - 1.476%) of the sorbed 137Cs from this muscovite test material. The Kd values calculated after the desorption of 137Cs were still generally large 6.93 x 103 mL/g to 1.40 x 106 mL/g. 137Cs was interpreted to be fixed at high affinity sites within the muscovite. This test material showed promise for being a sorbent for radiocesium contaminated waste solutions.

The protein SiaA (Streptococcal iron acquisition) is involved in heme uptake in the bacterium Streptococcus pyogenes. It is difficult to obtain this protein in its fully holo form (completely loaded with heme). To increase the concentration of heme in the growing cell, we added ä-aminolevulinic acid (ALA) and ferrous sulfate (FeSO4), precursors of heme, to the growth media. Neither increasing the concentration of heme in vivo, nor growth at lower temperature for longer times, increased the production of holoprotein. The classical method of measuring the concentration of heme in a newly discovered heme protein is cumbersome. We have developed an improved method, which gives a solution that is more stable and has a cleaner spectrum. With further development, this new technique may replace the classical assay. Background information on S. pyogenes, SiaA, ABC transporters, heme biosynthesis, and the pyridine hemochrome assay are described.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.