Department of Chemistry & Biochemistry

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1,2,3-Triazole-Heme Interactions in Cytochrome P450: Functionally Competent Triazole-Water-Heme Complexes
(American Chemical Society, 2012) Conner, Kip P.; Vennam, Preethi; Woods, Caleb M.; Krzyaniak, Matthew D.; Bowman, Michael K.; Atkins, William M.; University of Washington; University of Washington Seattle; University of Alabama Tuscaloosa
In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-alpha-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4 center dot 17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.
1-Propyl-4(5)-Methylimidazole Isomers for Temperature Swing Solvent Extraction
(MDPI, 2022) Qian, Shuai; Ward, Lauren M.; Rakers, Luke S.; Weinman, Steven T.; Bara, Jason E.; University of Alabama Tuscaloosa
Temperature swing solvent extraction (TSSE) utilizes an amine solvent with temperature-dependent water solubility to dissolve water at a lower temperature to concentrate or crystallize the brine and the phases are separated. Then, the water in solvent mixture is heated to reduce water solubility and cause phase separation between the solvent and water. The solvent and de-salted water phases are separated, and the regenerated solvent can be recycled. Issues with current TSSE solvents include the high solvent in water solubility and the high solvent volatility. This project used the highly tunable platform molecule imidazole to create two 1-butylimidazole isomers, specifically 1-propyl-4(5)-methylimidazole, to test their effectiveness for TSSE. The imidazoles take in more water than their current state-of-the-art counterparts, but do not desalinate the product water and dissolve in water at higher concentrations. Thus, while imidazoles make intriguing candidates for TSSE, further work is needed to understand how to design imidazoles that will be useful for TSSE applications.
3D Modeling of the Solidification Structure Evolution and of the Inter Layer/Track Voids Formation in Metallic Alloys Processed by Powder Bed Fusion Additive Manufacturing
(MDPI, 2022) Nastac, Laurentiu; University of Alabama Tuscaloosa
A fully transient discrete-source 3D Additive Manufacturing (AM) process model was coupled with a 3D stochastic solidification structure model to simulate the grain structure evolution quickly and efficiently in metallic alloys processed through Electron Beam Powder Bed Fusion (EBPBF) and Laser Powder Bed Fusion (LPBF) processes. The stochastic model was adapted to rapid solidification conditions of multicomponent alloys processed via multi-layer multi-track AM processes. The capabilities of the coupled model include studying the effects of process parameters (power input, speed, beam shape) and part geometry on solidification conditions and their impact on the resulting solidification structure and on the formation of inter layer/track voids. The multi-scale model assumes that the complex combination of the crystallographic requirements, isomorphism, epitaxy, changing direction of the melt pool motion and thermal gradient direction will produce the observed texture and grain morphology. Thus, grain size, morphology, and crystallographic orientation can be assessed, and the model can assist in achieving better control of the solidification microstructures and to establish trends in the solidification behavior in AM components. The coupled model was previously validated against single-layer laser remelting IN625 experiments performed and analyzed at National Institute of Standards and Technology (NIST) using LPBF systems. In this study, the model was applied to predict the solidification structure and inter layer/track voids formation in IN718 alloys processed by LPBF processes. This 3D modeling approach can also be used to predict the solidification structure of Ti-based alloys processes by EBPBF.
Abdominal segment reduction Development and evolution of a deeply fixed trait
(Landes Biocience, 2012) Yoder, John H.; University of Alabama Tuscaloosa
When a new student first begins to push flies, an immediate skill that must be learned is sorting the sexes. In Drosophila melanogaster several sexually dimorphic characters can be used to readily distinguish males from females including abdominal pigmentation, male sex combs and genital morphology. Another, often-overlooked, sexual dimorphism is adult abdominal segment number. Externally, adult Drosophila males possess one fewer abdominal segment than females; the terminal pregenital segment apparently either absent or fused with the next-most anterior segment. Beyond known roles for the homeotic protein Abdominal-B (Abd-B) and the sex-determining transcription factor Doublesex (Dsx) as key regulators of this trait, surprisingly little is known about either the morphogenetic processes or the downstream genetics responsible for patterning these events. We have explored both and found that rapid epithelial reorganization during pupation eliminates a nascent terminal male segment. We found this Abd-B-dependent process results from sex-and segment-specific regulation of diverse developmental targets including the wingless gene and surprisingly, dsx itself.(1,2) Here, I review our observations and discuss this trait as a model to explore both dynamics of epithelial morphogenesis as well as the evolution of developmental mechanisms.
Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid
(Portland Press, 2021) Chand, Subhash; Ray, Sriparna; Yadav, Poonam; Samanta, Susruta; Pierce, Brad S.; Perera, Roshan; University of Texas Arlington; University of Nebraska Medical Center; Creighton University; University of Alabama Tuscaloosa
To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E degrees = -260 mV) and mutant Mb (E degrees = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.
Accelerating the insertion reactions of (NHC)Cu-H via remote ligand functionalization
(Royal Society of Chemistry, 2021) Speelman, Amy L.; Tran, Ba L.; Erickson, Jeremy D.; Vasiliu, Monica; Dixon, David A.; Bullock, R. Morris; United States Department of Energy (DOE); Pacific Northwest National Laboratory; University of Alabama Tuscaloosa
Most ligand designs for reactions catalyzed by (NHC)Cu-H (NHC = N-heterocyclic carbene ligand) have focused on introducing steric bulk near the Cu center. Here, we evaluate the effect of remote ligand modification in a series of [(NHC)CuH](2) in which the para substituent (R) on the N-aryl groups of the NHC is Me, Et, Bu-t, OMe or Cl. Although the R group is distant (6 bonds away) from the reactive Cu center, the complexes have different spectroscopic signatures. Kinetics studies of the insertion of ketone, aldimine, alkyne, and unactivated alpha-olefin substrates reveal that Cu-H complexes with bulky or electron-rich R groups undergo faster substrate insertion. The predominant cause of this phenomenon is destabilization of the [(NHC)CuH](2) dimer relative to the (NHC)Cu-H monomer, resulting in faster formation of Cu-H monomer. These findings indicate that remote functionalization of NHCs is a compelling strategy for accelerating the rate of substrate insertion with Cu-H species.
Acetic Anhydride
(University of Alabama Libraries, 1929) Hanna, Carl; University of Alabama Tuscaloosa
Acetic anhydride is a colourless, volatile liquid having an odor like that of glacial acetic acid, but stronger. It is very corrosive and its vapors are extremely irritating to the eyes and lungs. Care should therefore be exercised in working with it, and any spilled upon the hands should be immediately washed off. It is a condensation of two molecules of acetic acid with elimination of water. The reaction, however, is reversible, and there is always present some acetic acid due to action of water.
Achieving Excellence in Graduate Research: A Guide for New Graduate Students
(Wiley-Blackwell, 2015) Parker, Charles B.; Amsden, Jason J.; Peng, Qing; Stoner, Brian R.; Glass, Jeffrey T.; Duke University; University of Alabama Tuscaloosa; Research Triangle Institute
Acyclovir as an Ionic Liquid Cation or Anion Can Improve Aqueous Solubility
(American Chemical Society, 2017) Shamshina, Julia L.; Cojocaru, O. Andreea; Kelley, Steven P.; Bica, Katharina; Wallace, Sergey P.; Gurau, Gabriela; Rogers, Robin D.; University of Alabama Tuscaloosa; McGill University; Technische Universitat Wien; Tennessee Technological University
Six ionic liquid (IL)-forming ions (choline, tetrabutylphosphonium, tetrabutylammonium, and trimethyl-hexadecylammonium cations, and chloride and docusate anions) were paired with acyclovir as the counterion to form four low melting solid salts and two waxes; five of these compounds could be classified as ILs. All of the newly synthesized acyclovir ILs exhibited increased aqueous solubilities by at least 2 orders of magnitude when compared to that of neutral acyclovir. For three of the prepared compounds, the solubilities in simulated body fluids (phosphate-buffered saline, simulated gastric, and simulated intestinal fluids) were also greatly enhanced when compared to that of neutral acyclovir. Acyclovir in its anionic form was more water-or buffer-soluble than acyclovir in its cationic form, though this might be the effect of the particular ions, indicating that the solubilities can be finely tuned by proper choice of the cationic or anionic form of acyclovir and the counterion paired with it.
Air-stable palladium(II) precatalysts: synthesis, properties, and applications in cross-coupling reactions
(University of Alabama Libraries, 2017) Barnett, Kerry Lynn; Shaughnessy, Kevin H.; University of Alabama Tuscaloosa
Palladium catalyzed cross-coupling reactions are a versatile tool in organic chemistry to produce small molecules. Current research interests involve the development of new catalyst systems for various palladium catalyzed reactions. Previous work has suggested the active species for palladium-catalyzed cross-coupling reactions to be a mono-ligated palladium(0) species when sterically demanding ligands are used. Therefore, the my work focused on the synthesis of precatalysts with an established 1:1 L:Pd. Specifically, we were focused on the synthesis of mono-ligated palladium(II) precatalysts of general formula [(R_3P)PdCl_2]_2, using di-tert-butylneopentylphosphine (DTBNpP), tert-butyldineopentylphosphine (TBDNpP), and trineopentylphosphine (TNpP) as ligands. Under optimized conditions, both [(DTBNpP)PdCl_2]_2 and [(TNpP)PdCl_2]_2 were effective precatalysts for the Suzuki cross-coupling of a wide range of aryl bromides. Comparison studies with the air-stable precatalysts versus the in-situ generated catalyst showed the precatalysts to have improved conversions and higher rates of reaction under both inert and ambient atmospheres. The precatalyst activation pathways were investigated by 31P NMR spectroscopy. The spectra obtained from 31P NMR experiments revealed a side reaction resulting in a catalytically inactive palladacycle species. The results obtained from the mechanistic investigations led to further optimization of reaction conditions to decrease the amounts of catalytically inactive side products formed.
Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons
(MDPI, 2020) Park, Han-A; Mnatsakanyan, Nelli; Broman, Katheryn; Davis, Abigail U.; May, Jordan; Licznerski, Pawel; Crowe-White, Kristi M.; Lackey, Kimberly H.; Jonas, Elizabeth A.; University of Alabama Tuscaloosa; Yale University
B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to N-Bcl-xL, and accumulation of N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as alpha-tocotrienol (TCT) will prevent N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with alpha-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and N-Bcl-xL levels. N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. alpha-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing N-Bcl-xL, suggesting that N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.
The Anionic Polymerization of a tert-Butyl-Carboxylate-Activated Aziridine
(MDPI, 2022) Giri, Chandan; Chang, Jen-Yu; Mbarushimana, Pierre Canisius; Rupar, Paul A.; University of Alabama Tuscaloosa
N-Sulfonyl-activated aziridines are known to undergo anionic-ring-opening polymerizations (AROP) to form polysulfonyllaziridines. However, the post-polymerization deprotection of the sulfonyl groups from polysulfonyllaziridines remains challenging. In this report, the polymerization of tert-butyl aziridine-1-carboxylate (BocAz) is reported. BocAz has an electron-withdrawing tert-butyloxycarbonyl (BOC) group on the aziridine nitrogen. The BOC group activates the aziridine for AROP and allows the synthesis of low-molecular-weight poly(BocAz) chains. A C-13 NMR spectroscopic analysis of poly(BocAz) suggested that the polymer is linear. The attainable molecular weight of poly(BocAz) is limited by the poor solubility of poly(BocAz) in AROP-compatible solvents. The deprotection of poly(BocAz) using trifluoroacetic acid (TFA) cleanly produces linear polyethyleneimine. Overall, these results suggest that carbonyl groups, such as BOC, can play a larger role in the in the activation of aziridines in anionic polymerization and in the synthesis of polyimines.
Anionic polymerization of activated aziridines
(University of Alabama Libraries, 2018) Mbarushimana, Pierre Canisius C.; Rupar, Paul A.; University of Alabama Tuscaloosa
Polyethyleneimine, also referred to as polyaziridine, is a polymer that has a repeat unit of (-CH2CH2NH-). Due to its high amine density, PEI finds application in several domains including gene transfection, antimicrobial/antibacterial medicine, CO2 capture, thin film metal deposition, industrial wastewater treatments, and more. Commercially, PEI is obtained via the uncontrolled polymerization of aziridine and the cationic ring opening polymerization (CROP) of oxazolines. However, the available cationic polymerization techniques of aziridines are poorly controlled. In order to synthesize well-defined PEIs, recent literature approaches have used the anionic ring opening polymerization (AROP) of various N-substituted aziridines. The present research, done at The University of Alabama, has focused on the synthesis and AROP of N-substituted aziridines, without the substitution at the 2-position of the aziridine ring. The studied monomers include three tert-butylcarboxy-protected aziridine monomers, specifically tert-butyl aziridine-1-carboxylate, tert-butyl 2-methylaziridine-1-carboxylate, tert-butyl 2-decylaziridine, and six 1-(alkylsulfonylsulfonyl) aziridine monomers, i.e. 1-(methylsulfonyl)aziridine, 1-(toluenesulfonyl)aziridine, 1-(octylsulfonyl)aziridine, 1-(sec-butylsulfonyl)aziridine, 1-((2-nitrophenyl)sulfonyl)aziridine, and 1-((4-nitrophenyl)sulfonyl)aziridine. After characterization, the synthesized monomers were homopolymerized by AROP using various nucleophilic initiators. Except for BOCDecAz, all the synthesized monomers were successfully converted into the corresponding polymers. However, only short oligomers of the resulting homopolymers were formed, as high molecular weight polymer chains were insoluble. The synthesized polymers can potentially be used as precursor to pure linear polyamines A single sulfonyl aziridine, namely1-((ortho-nitrophenyl)sulfonyl)aziridine (oNsAz) was found to produce high molecular weight poly(oNsAz) that was soluble in DMF and DMSO. This is significant as it is the first example of a soluble poly(1-sulfonylaziridine) homopolymer; prior examples were limited to random copolymer. The deprotection of poly(oNsAz) was also attempted in effort to synthesize linear PEI. Although evidence was found for the formation of linear PEI, satisfactory purification of the linear PEI was not achievable.
The anionic ring-opening polymerization of cyclic imines
(University of Alabama Libraries, 2019) Reisman, Louis; Rupar, Paul A.; University of Alabama Tuscaloosa
One important class of polymers is polyimines. Polyimines have a wide range of applications such as CO2 capture and non-viral gene-transfection. Despite their many applications, the use of these polymers is limited due to difficulties in controlling the polymerization. To increase the feasibility of polyimines in the aforementioned high-value applications, the research in this dissertation focuses on controlled routes to produce linear polyimines using living anionic ring-opening polymerization (AROP). The early work of this dissertation focuses on an AROP route to linear polyethylenimine (LPEI). Due to the insolubility of p(N-sulfonylaziridine)s (i.e. non-2-substituted) in all common solvents, this was achieved using a copolymerization strategy. Utilizing two N-sulfonylaziridines, N-(methanesulfonyl)aziridine (MsAz) and N-(sec-butylsulfonyl)aziridine (sBsAz), with similar reactivities, a soluble random copolymer with narrow molecular weight distributions is produced. Removal of the sulfonyl groups of p(MsAz-r-sBsAz) affords the first example of LPEI by living, controlled AROP. The later work in this dissertation focuses on the AROP of N-sulfonylazetidines in route to linear poly(trimethylenimine) (LPTMI). Initially, the polymerization of N-(methanesulfonyl)azetidine (MsAzet) was investigated. The kinetics of this polymerization were studied, and the reaction found to be first order with respect to monomer and the number of active chain ends remains constant throughout the polymerization. Interestingly, activation occurs at the methanesulfonyl group, leading to polymer branching. This branching precludes p(MsAzet) from being a precursor to LPTMI. By more judicial selection of N-sulfonylazetidine monomers, a living, controlled AROP approach to LPTMI was achieved by copolymerizing two similar N-sulfonylazetidines, N-(p-tolylsulfonyl)azetidine (pTsAzet) and N-(o-tolylsulfonyl)azetidine (oTsAzet), to produce a statistical copolymer. Copolymerization was required as the homopolymers resulting from these monomers were insoluble in all common solvents. The copolymerization is living and controlled, producing polymers with narrow molecular weight distributions. The kinetics of the copolymerization, and the reactivity ratios of the two monomers, were studied and the sulfonyl groups of the polymer were removed to provide the first example of LPTMI by living, controlled AROP. Finally, the high barrier to polymerization of N-sulfonylazetidines was utilized to produce block copolymers, containing no homopolymer impurities, in a closed system in which all monomers are present in solution at the time of initiation.
Anti-Apoptotic Effects of Carotenoids in Neurodegeneration
(MDPI, 2020) Park, Han-A; Hayden, Mary Margaret; Bannerman, Sydni; Jansen, Joseph; Crowe-White, Kristi M.; University of Alabama Tuscaloosa
Apoptosis, programmed cell death type I, is a critical part of neurodegeneration in cerebral ischemia, Parkinson's, and Alzheimer's disease. Apoptosis begins with activation of pro-death proteins Bax and Bak, release of cytochrome c and activation of caspases, loss of membrane integrity of intracellular organelles, and ultimately cell death. Approaches that block apoptotic pathways may prevent or delay neurodegenerative processes. Carotenoids are a group of pigments found in fruits, vegetables, and seaweeds that possess antioxidant properties. Over the last several decades, an increasing number of studies have demonstrated a protective role of carotenoids in neurodegenerative disease. In this review, we describe functions of commonly consumed carotenoids including lycopene, beta-carotene, lutein, astaxanthin, and fucoxanthin and their roles in neurodegenerative disease models. We also discuss the underlying cellular mechanisms of carotenoid-mediated neuroprotection, including their antioxidant properties, role as signaling molecules, and as gene regulators that alleviate apoptosis-associated brain cell death.
Antioxidant Activity in Supramolecular Carotenoid Complexes Favored by Nonpolar Environment and Disfavored by Hydrogen Bonding
(MDPI, 2020) Gao, Yunlong; Focsan, A. Ligia; Kispert, Lowell D.; Nanjing Agricultural University; University System of Georgia; Valdosta State University; University of Alabama Tuscaloosa
Carotenoids are well-known antioxidants. They have the ability to quench singlet oxygen and scavenge toxic free radicals preventing or reducing damage to living cells. We have found that carotenoids exhibit scavenging ability towards free radicals that increases nearly exponentially with increasing the carotenoid oxidation potential. With the oxidation potential being an important parameter in predicting antioxidant activity, we focus here on the different factors affecting it. This paper examines how the chain length and donor/acceptor substituents of carotenoids affect their oxidation potentials but, most importantly, presents the recent progress on the effect of polarity of the environment and orientation of the carotenoids on the oxidation potential in supramolecular complexes. The oxidation potential of a carotenoid in a nonpolar environment was found to be higher than in a polar environment. Moreover, in order to increase the photostability of the carotenoids in supramolecular complexes, a nonpolar environment is desired and the formation of hydrogen bonds should be avoided.
Application of hydrogen deuterium exchange mass spectrometry in protein-ligand and protein-protein interactions
(University of Alabama Libraries, 2016) Guan, Siqi; Frantom, Patrick A.; University of Alabama Tuscaloosa
Proteins are not static objects. They have a great variety of internal motions with different amplitudes and different timescales. These internal motions play an important role in catalytic processes. Therefore, the existence of an intimate relationship between protein dynamics and protein function is widely accepted. Due to the significance of protein dynamics, techniques have been developed to study protein dynamics including nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and mass spectrometry (MS). Compared with NMR and EPR spectroscopy, MS has less stringent sample requirements, including protein concentration and protein size. Moreover, the mass accuracy, sensitivity, and faster data analysis also have contributed to the rapid growth of MS based techniques. Hydrogen-deuterium exchange mass spectrometry (HDX-MS), a combination of HPLC and MS, has become a common and sensitive tool to probe protein structural flexibility and solution dynamics. In this dissertation, HDX-MS was applied to study dynamic changes of proteins due to substrate binding and protein-protein interactions. The GT-A glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis (MtGpgS) catalyzes the first step of biosynthesis of 6-O-methylglucose lipopolysaccharides (MGLPs), which are essential to growth and existence of mycobacterium. The HDX-MS data revealed that the two substrates UDP-glucose (UDPG) and 3-phosphoglycerate (3PGA) can bind to MtGpgS independently, disagreeing with the previous proposal that 3PGA can only bind to MtGpgS after UDPG. Moreover, 3PGA was found to bind to or allosterically affect the UDPG binding site. Furthermore, the HDX-MS data revealed that MtGpgS may provide a necessary conformation for UDPG binding, while it goes through a large conformational change on 3PGA binding. The GT-B glycosyltransferase MshA from Corynebacterium glutamicum (CgMshA) catalyzes the initial step of mycothiol biosynthesis. A large conformational change was observed in CgMshA on nucleotide binding by superimposing APO structure of CgMshA and complex structure with UDP. HDX-MS was utilized to study conformational changes of CgMshA on substrate binding from the aspect of dynamics, providing a complementary to static structures. The HDX-MS data showed that both substrates uridine diphosphate glucose-N-acetylglucosamine (UDP-GlcNAc) and 1-L-myo-inositol-1-phosphate (I1P) can bind to CgMshA independently, but the I1P binding is not productive since it binds to an uncorrect site. Moreover, the I1P binding can lead to dynamic changes of CgMshA, while only UDP-GlcNAc can induce the major conformational change of CgMshA. Furthermore, the 3PGA binding cannot induce further dynamic changes of CgMshA in the presence of UDP. HDX-MS was also employed to study dynamic changes of protein complex SufBC2D from Escherichia coli on ADP/Mg2+ binding. This complex is responsible for Fe-S cluster assembly under oxidative stress. The crystal structure of SufBC2D complex has been determined, while little dynamic information is known. So HDX-MS was applied to study dynamic changes of the SufBC2D complex. The HDX-MS data revealed that SufC has a significant conformational change, which may be required by ATP binding and hydrolysis. Moreover, SufB and SufD are detected to have dynamic changes due to SufC conformational changes. These dynamic changes suggest that SufB-SufD protomer may have a conformational change in order to provide a suitable conformation for Fe-S cluster assembly. This work demonstrates that HDX-MS can be effectively used to study protein-ligand and protein-protein interactions, as well as the accompanying changes in structural dynamics. HDX-MS data detects substrate binding mechanism and conformational changes that are not available through x-ray crystallography. With these advantages, HDX-MS has been applied in study of protein structure and dynamics, studying protein-ligand and protein-protein interactions, protein folding, as well as protein therapeutics discovery and development.
Applications of polyamidoamine dendrimers in polymer electrolyte membrane fuel cells
(University of Alabama Libraries, 2009) Zhu, Huizhen; Thrasher, Joseph S.; University of Alabama Tuscaloosa
Dendrimers are highly branched macromolecules with well-ordered three- dimensional architectures. Polyamidoamine (PAMAM), the most common class of dendrimers, have been widely studied due primarily to the following three features: 1) the interior amine and amide groups that can interact with ionic metal precursors through ligand exchange reactions; 2) the presence of an interior void space in the higher generation dendrimers; and 3) the exterior primary amine groups that permit further functionaliztion. These unique structural features have inspired many potential applications. This dissertation describes two applications of PAMAM dendrimers in polymer electrolyte membrane fuel cells (PEMFCs). First, in an effort to improve the utility of Pt in PEMFCs, PAMAM G4 was used as both a template and a stabilizer to synthesize dendrimer encapsulated Pt nanoparticles (Pt DENs) by photoreduction. These nanoparticles are highly monodisperse, exhibit high specific activity for the oxygen reduction reaction, and are inert to methanol oxidation, showing great potential for application in PEMFCs. Then, a simplified membrane electrode assembly (MEA) has been fabricated by the electrostatic self-assembly between Nafion® and Pt DENs and characterized. Two methods were proposed to increase Pt loading: layer-by-layer self-assembly and immobilization of Pt DENs and carbon powder on carbon fibers. Approximately 80 layers were proposed to reach the required loading using a dipping machine. Immobilization of Pt DENs and carbon powder simultaneously on carbon fibers can easily be achieved by electrochemical coupling, which is promising for replacing the conventional method of electrode fabrication. Secondly, in order to reduce the methanol crossover in direct methanol fuel cells (DMFCs), PAMAM G0 doped Nafion® membranes were prepared. Direct TEM imaging of the Naifon® embedded with nanoparticles demonstrates that PAMAM G0 can penetrate into the bulk of Nafion® through cluster channels to re-organize the distribution of sulfonate clusters by interacting with the sulfonic acid groups in different clusters. The presence of PAMAM G0 in the Nafion® membrane causes reduction of both methanol permeability and proton conductivity, but a very beneficial trade off can be reached when a doping concentration of 10⁻⁴ M PAMAM G0 is used. The fuel cell performance is much improved When Nafion® was treated with 10⁻⁴ M PAMAM G0.
Approaches to the Synthesis of Qinghaosu
(University of Alabama Libraries, 1984) Muse, David Earl; University of Alabama Tuscaloosa
The object of this research was to develop new syntheses of bicyclic peroxides and to apply these new methods to the total synthesis of qinghaosu (Arteannuin A), a previously reported anti-malarial compound. 4 Qinghaosu has shown clinical activity in humans against chloroquineresistant strains of malaria. The most challenging feature of qinghaosu, and the biologically active portion of the compound was the peroxide bridge.
Arabidopsis Toxicos en Levadura 12 Modulates Salt Stress and ABA Responses in Arabidopsis thaliana
(MDPI, 2022) Kong, Feng; Ramonell, Katrina M.; University of Alabama Tuscaloosa; University of Georgia
Salt is one of the most common abiotic stresses, causing ionic and osmotic pressure changes that affect plant growth and development. In this work, we present molecular and genetic evidence that Arabidopsis Toxicos en Levadura 12 (ATL12) is involved in both salt stress and in the abscisic acid response to this stress. We demonstrate that ATL12 is highly induced in response to salt stress and that atl12 mutants have a lower germination rate, decreased root length, and lower survival rate compared to the Col-0 wild-type in response to salt stress. Overexpression of ATL12 increases expression of the salt stress-associated genes SOS1/2, and ABA-responsive gene RD29B. Additionally, higher levels of reactive oxygen species are detected when ATL12 is overexpressed, and qRT-PCR showed that ATL12 is involved in the AtRBOHD/F-mediated signaling. ATL12 expression is also highly induced by ABA treatment. Mutants of atl12 are hypersensitive to ABA and have a shorter root length. A decrease in water loss and reduced stomatal aperture were also observed in atl12 mutants in response to ABA. ABA-responsive genes RD29B and RAB18 were downregulated in atl12 mutants but were upregulated in the overexpression line of ATL12 in response to ABA. Taken together our results suggest that ATL12 modulates the response to salt stress and is involved in the ABA signaling pathway in Arabidopsis thaliana.

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