Chemistry

Permanent URI for this collectionhttps://hdl.handle.net/10323/11885

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Disease mechanisms of spectrin superfamily proteins
(2024-01-01) Atang, Alexandra E; Avery, Adam W; Wu, Colin G; Delorme-Axford, Elizabeth
Spectrin superfamily proteins are a group of evolutionarily linked proteins that share in actin crosslinking functions. Characterized by an N-terminal actin binding domain (ABD), comprised of tandem calponin homology (CH) domains, a variable number of spectrin repeat domains, and in some cases an EF hand motif, these cytoskeletal proteins are an integral feature of the submembranous cellular structure. Mutations within the ACTN2 and SPTBN2 genes that encode α-actinin-2 and β-III-spectrin, respectively are known to cause disease. Predominately expressed in cardiomyocytes of the heart, α-actinin-2 mutations localizing to the ABD are linked to cardiomyopathy, leading to heart failure and sudden cardiac death. ABD localized β-III-spectrin mutations cause the rare neurodegenerative disease, spinocerebellar ataxia type 5 (SCA5). Previous studies of a L253P mutation within the β-III-spectrin ABD revealed an increased actin-binding affinity. Positioning of the L253P mutation to the interface of CH1 and CH2 was demonstrated to promote a conformational opening, leading to high affinity actin interactions. These studies investigate the molecular consequences of nine additional β-III-spectrin ABD-localized, SCA5 missense mutations (V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R) and three α-actinin-2 ABD-localized, cardiomyopathy missense mutations (A119T, M228T, and T247M). Importantly, all β-III-spectrin and α-actinin-2 mutations localizing the CH1-CH2 interface increased actin-binding affinity, while the actin-binding surface localized α-actinin-2 mutation, A119T, decreased actin-binding affinity. Using biochemical and biophysical approaches, we demonstrate that the mutant β-III-spectrin and α-actinin-2 ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all ABD mutations are destabilizing, suggesting structural disruptions to the ABDs. Additional in vivo studies of the β-III-spectrin mutations indicate an accumulation of protein within neurons, likely a result of increased actin binding-affinity. Altogether, the data indicate aberrant actin-binding affinity is a shared molecular consequence of ABD localized mutations of spectrin superfamily proteins.
Organic Geochemistry in Oceanic Hydrothermal Systems: Implications for Prebiotic Synthesis, Extraterrestrial Habitability, and Green Chemistry
(2025-01-01) Aspin, Alexandria Rose; Yang, Ziming; Beyeh, Ngong; Chavez, Ferman A
Hydrothermal systems are unique environments that are relevant to manydifferent applications. In particular, this dissertation details their relevance to the origin of life on Earth, the search for extraterrestrial habitable environments, and their use in sustainable practices. Chapters Two and Three seek to investigate the influences of parameters such as temperature, pH, and metal salts on the reaction pathways of important organics, including alkenes and amino acids, respectively. Acidic solutions promote the hydration and dimerization pathways of alkenes, while the presence of metal salts can enhance the dimerization and oxidation pathways. Similarly, these geochemical parameters influence amino acid reaction pathways and product distributions, where lower temperatures, basic pHs, and inorganic metals inhibit their degradation. Chapter Three indicates the enhanced survivability of amino acids in environments that are expected on icy ocean moons, suggesting they may be prime targets for the search for life outside of Earth. Chapter 4 investigates whether the transition from liquid in the subsurface ocean to vacuum above the surface influences the relative abundances of biosignatures, as future missions will rely on the analysis of materials above the icy surfaces to determine whether they could be inhabited. Simulation of the liquid-vacuum transition indicated the distributions were sufficiently conserved to allow distinction between biological and abiotic sources. Finally, the green and sustainable recycling of plastic using Earth-abundant minerals has been investigated under hydrothermal conditions. The efficiency of plastic degradation was strongly influenced by its structure, where polymers with a C-C backbone degraded much slower under hydrothermal treatment compared to polymers containing a heteroatomic backbone. Furthermore, the presence of minerals selectively enhanced the degradation of heteroatomic plastics, highlighting the application of hydrothermal conversion as a green and sustainable alternative for pollutant recycling.
Mechanisms of G-quadruplex Recognition and Unfolding by the FANCJ Helicase and REV1 Polymerase
(2024-01-01) Lowran, Kaitlin Antha; Wu, Colin G; Avery, Adam; Villa-Diaz, Luis G
G-quadruplexes (G4s) are secondary structures formed by guanine-rich nucleic acid sequences. Accumulated G4s in cells can disrupt DNA replication, RNA transcription, and other cellular processes. G4 DNA can adopt parallel, antiparallel, or hybrid conformations depending on solution conditions and base sequence. Moreover, reactive oxygen species can readily convert guanine to 8-oxoguanine (8oxoG) to form 8oxoG4s. However, the precise mechanisms by which repair proteins recognize and remove such lesions remain unclear. The FANCJ DNA helicase possesses an AKKQ motif that functions as a G4-targeting site that binds to G4s and 8oxoG4s. Additionally, FANCJ interacts with the REV1 translesion synthesis polymerase to facilitate replication across G4-forming sites. Although REV1 can also target and disrupt G4s, it is unknown whether the activities of FANCJ and REV1 are redundant or if they act on specific G4 substrates. We hypothesized that the presence of 8oxoG would impact the folding and stability of parallel, antiparallel, and hybrid G4s in specific positions within the G4. Furthermore, we anticipated that FANCJ and REV1 would have distinct 8oxoG4 binding preferences that can be correlated with the physical properties of the 8oxoG4s. We predicted that the collaborative action of FANCJ and REV1 would repair tightly-folded 8oxoG4 structures, whereas REV1 alone may suffice for disrupting the loosely folded 8oxoG4s. Our results revealed that introducing 8oxo1 significantly reduced the thermal stability of both parallel and antiparallel G4 structures. While 8oxo5 destabilized hybrid structures. FANCJ and REV1 both preferred parallel G4 and 8oxoG4s due to their greater stability and tighter folding compared to hybrid or antiparallel conformations. Furthermore, FANCJ had the lowest affinity for antiparallel 8oxo3 G4s, while REV1 had a lower affinity for hybrid 8oxo1 G4s. These findings indicate that the presence of 8oxoG modification in G4 structures may hinder the binding of these proteins. The study revealed that both FANCJ and REV1 have distinct substrate-specific preferences for unfolding G4s. FANCJ demonstrated lower unfolding rates when 8oxo5 was present within the G4 stack, indicating that the position of the lesion plays a critical role in the unfolding efficiency. REV1, on the other hand, showed a lower unfolding rate for more stable G4 structures like the GGGT G4, but faster rates for the oxidized constructs. This research provides significant insights into the specific roles and mechanisms of FANCJ and REV1 in unfolding 8oxoG-damaged G4s, contributing to our understanding of DNA repair mechanisms.
Investigation of Different Organic Functional Groups Transformations in Laboratory-Simulated Hydrothermal Systems
(2023-01-01) Liao, Yiju; Yang, Ziming; Zeng, Xiangqun; Beyeh, Ngong; Bianchette, Thomas
The study of organic reactions within natural hydrothermal environments is significant for our understanding of the profound carbon and nitrogen cycles, the life dynamics of deep microbial populations, and possibly, lifes origins. Numerous reaction routes that involve organic compounds under geochemically pertinent hydrothermal conditions have been identified. However, the underlying mechanisms of these reactions, particularly those associated with inorganic substances, remain largely unexplored. This research aims to demystify these mechanisms to enable the development of predictive reactivity models and the investigation of potential applications of these reactions beyond the field of geochemistry. The focus of this dissertation is the mechanisms of interconversion of alcohols, aldehydes, amines with and without the presence of metal ions. Dehydration is always the primary pathway for alcohols in the hydrothermal fluids. However, in the presence of copper(II) or iron(III) salts, the oxidation of alcohols is greatly promoted and becomes a competitive pathway to form aldehydes and carboxylic acids as the major products. Inspired by the role of iron ions, we reported the first examples of oxidation of aldehydes with Fe(NO3)3 in water under anaerobic hydrothermal conditions in green and relatively clean way, with relatively high yields of carboxylic acids achieved within hours. A framework was investigated for selecting amines as geochemical tracers based on kinetic modeling. Even though the catalysis from minerals can be neglectable, the presence of metal ions altered the pathway by aerobic oxidation reaction. The geomimetic reactions discussed in this dissertation could potentially be employed in biomass oxidation to yield beneficial fuels and other high-value chemicals. Leveraging plentifully available metal ions and water as the solvent, these reactions offer a sustainable alternative to the existing biomass oxidation methods. The latter often involve the use of rare, exotic metal catalysts and organic solvents, presenting an environmental challenge. Thus, the exploration of these environmentally friendly reactions provides a greener approach to pollutants oxidation.
Assessment of Frequency, Degradation, Normalization, Inhibition, and Potential Surrogates of the Sars-Cov-2 Gene Targets
(2023-01-01) Hunawill, Emily; Szlag, David; Wu, Colin; Avery, Adam; Wendell, Doug; Westrick, Randal
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Our study focuses on identifying and quantifying the presence of SARS-CoV-2 in the sewage based on identifying three gene targets which target the nucleocapsid gene (N1 and N2) and the envelope gene (E). The first set of experiments looked at the degradation of these targets at 4C, 25C, and 35C. As temperature increased so did the rate constants. Based on the half-life data, N1 degraded faster than N2 at 4C, all targets degraded at the same rate at 25C, and E degraded faster than N1 and N2 at 35C. Frequency of the gene targets was then assessed. E occurred less frequently than N1 and N2 adding 2.3 more SARS-CoV-2 detections leading to its removal from testing. The N1 and N2 gene targets were both necessary with removal of one resulting in minimally 9.6 loss of SARS-CoV-2 detections. We investigated normalization of the N1 and N2 gene targets with four different fecal indicators (pepper mottled mild virus, HF183, lachno3, and crAssphage) to improve the correlation between the sewage signal and clinical cases. Normalizing the data did not result in an increased correlation between the clinical and sewage data. Bovine coronavirus (BCoV) and Phi6 viruses were evaluated as surrogates to estimate SARS-CoV-2 inhibition in a single duplex reaction. Duplexing these targets was successful without significant signal loss, and these targets were used to estimate SARS-CoV-2 inhibition. Compared to BCoV, Phi6 suggested over 10 more fully inhibited samples. Several workflow modifications including bovine serum albumin (BSA), dilution, polyvinylpyrrolidone, and pasteurization were applied to the sewage samples in an attempt to reduce sample inhibition. BSA was able to reduce inhibition for both N1 and N2. Lastly, BCoV, Phi6, and murine hepatitis virus (MHV) retrieval were compared to the retrieval of the SARS-CoV-2 gene targets in sewage to determine if they accurately depicted the amount of inhibition the SARS-CoV-2 targets exhibited. BCoV and MHV were better surrogates to assess SARS-CoV-2 inhibition. SARS-CoV-2 RNA concentration in sewage were shown to correlate with COVID-19 cases.
Benzodiazepine Coordination Chemistry And Nitrogen Heterocyclic Compounds From Reactions Of Carbonyl Alkynes With O-Phenylenediamines
(2022-07-19) Twardy, Dylan Joseph; Dembinski, Roman; Beyeh, Ngong; Chavez, Ferman; Wheeler, Kraig; Yang, Ziming
The presence of heterocyclic compounds in active pharmaceutical ingredients and natural products implicates their importance to synthetic chemistry. Moreover, their inherent structures offer potential as metal-chelators. This work involved the design of simple methods for the construction of new nitrogen-containing heterocycles and to explore examples of coordination complexes. Benzodiazepines and their derivatives are biologically active heterocycles often prescribed as a treatment for anxiety, epilepsy, and insomnia. In addition, benzimidazo[2,1-a]isoquinolines are another class of biologically active heterocycles that are composed of moieties inherent to a wide variety of active pharmaceutical ingredients. Herein, the microwave-assisted reaction in ethanol of o-phenylenediamines with either alk-2-ynones or 2-ethynyl benzaldehydes was found to yield 1,5-benzodiazepines and benzimidazo[2,1-a]isoquinolines, respectively. To facilitate selective coordination of benzodiazepines, new pyridine containing 1,5-benzodiazepine chelators were synthesized and combined with metal reagents to form new benzodiazepine metal complexes characterized by X-ray crystallography.

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