Chemistry & Biochemistry Doctoral Work

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Structural Basis of Key Conserved Cell-Surface Signaling Pathways Involved in Iron Import
(North Dakota State University, 2024) Jernberg, Beau
Cell surface signaling (CSS) allows Gram-negative bacteria to transcriptionally regulate gene expression in response to external stimuli. CSS pathways involve three key components: an outer membrane transducer for sensing the extracellular stimuli; an inner membrane sigma regulator for relaying the signal; and a cytoplasmic sigma factor, which activates transcription of target genes. The goal of this research was to structurally and biophysically characterize events leading to the processing of the sigma regulator that results in transcription activation. Our model systems are the Pseudomonas capeferrum BN7/8 (Pup) and Escherichia coli ferric citrate (Fec) uptake pathways. We detail the X-ray crystal structure of the N-terminal signaling domain (NTSD) of the transducer, PupB, complexed with the C-terminal cell-surface signaling domain (CCSSD) of the sigma regulator, PupR. Stabilization of the PupR CCSSD by the PupB NTSD provides a rationale for the formation of a preformed CSS complex. Additionally, we probed the FecR CCSSD and FecA NTSD interaction and observed similarities. We found the FecA NTSD complexes with the FecR CCSSD and stabilizes the domain in nonsignaling conditions indicating a conserved mechanism. Further, we show that access to the PupR CCSSD is only possible in the absence of the PupB NTSD. Pulldown assays, isothermal titration calorimetry, protease assays, and mass spectrometry analysis demonstrate the site-1 protease, Prc, only recognizes and degrades PupR in the absence of the PupB NTSD. X-ray crystal structures of Prc mutants and potential product peptides reveal transitions between “closed” and “open” conformations as well as catalytic intermediates in the protease active site. Size exclusion small angle X-ray scattering data confirms the Prc conformations in solution and an elongated molecular envelope of the Prc:PupR complex. Together this provides new structural insights into protease activation during CSS. Finally, we studied the TonB C-terminal domain of P. capeferrum by size exclusion small angle X-ray scattering. Our results indicate it forms a monomeric structure in solution. Overall, our results indicate there is a conserved CSS pathway that has been characterized by our individual signal transduction states. Thus, we have provided novel implications in ferric siderophore uptake and the mechanism of iron import mediated CSS.
Investigating the role of BECN2 CCD interactions in canonical and non-canonical autophagy
(North Dakota State University, 2024) Bueno, Elizabeth
Autophagy is a conserved cell-survival pathway wherein old, damaged or harmful cellular components are surrounded by a double membrane vesicle called the autophagosome for lysosomal degradation and recycling. All eukaryotes have a conserved BECN homolog, a key coiled-coil domain (CCD)-containing autophagy protein. Mammals are unique as they have two BECN homologs, BECN1 and BECN2, both of which have the same domain architecture and function in autophagy. BECN2 has been shown to also function in non-canonical autophagy. My research focused primarily on investigating selected interactions of the BECN2 CCD. We investigated interactions of the BECN2 CCD with two CCD-containing proteins known to bind to BECN1: UVRAG, an important autophagy protein, and TAB2, a protein important for inflammatory responses. We show that the BECN2 and UVRAG CCDs interact, but were unable to purify stable complexes for structural studies. In comparison, the CCDs of BECN2 and TAB2 bind with an affinity tighter than that of BECN2 homodimerization, forming a well-folded elongated heterodimer. These preliminary results provide information regarding interactions that enable BECN2 to regulate autophagy, in a manner analogous to BECN1. We also show that, unlike BECN1, BECN2 facilitates non-canonical autophagy involving ATG9A-bearing vesicles, via interactions with the STX6 SNARE domain. We show that the STX6 SNARE domain binds to BECN2 residues 181-250 (BECN2(181-250)) within the BECN2 CCD. The STX6 SNARE is disordered in the absence of interacting partners but becomes helical when in complex with BECN2. The BECN2(181-250):STX6 SNARE complex is also more stable than either the BECN2(181-250) or the BECN2 CCD homodimer. We determined the 2.65 Å X-ray crystal structure of the STX6 SNARE bound to BECN2, showing that the complex is a heterotrimeric helical bundle, consisting of one helix comprising BECN2(181-250) and two STX6 SNAREs that are anti-parallel to each other. The heterotrimer interface is stabilized by 15 layers of three residues, each contributed by a different helix, of which, six layers are entirely hydrophobic, including two consisting of three leucines each. We verified the importance of these hydrophobic layers using point mutations and affinity pulldowns, showing that mutations of the hydrophobic layers either significantly or completely disrupt the interaction between BECN2(181-250) and STX6 SNARE domain. This 3-helix bundle likely represents an intermediate during the formation of the full SNARE complex, thereby providing mechanistic insights into the process by which ATG9A-bearing vesicles transport lipids to the growing phagophore. Together these studies help explain the role of the BECN2 CCD in canonical and non-canonical autophagy, providing clues as to why mammals have two BECN paralogs.
Synthesis and Characterization of Thieno[3,4-b]pyrazine-Based Near-Infrared Material
(North Dakota State University, 2024) Gilman, Spencer
Since their first report in the 1800s, conjugated polymers have gained significant attention for their ability to exhibit the optical and electronic properties of inorganic semiconductors and the physical traits of organic plastics. This has led to the development of organic electronics with notable commercial applications, such as organic photovoltaics and organic light-emitting diodes. The tunability of these materials has also allowed for the production of materials that absorb and emit near-infrared (NIR) light, making them useful for NIR photodetection and bioimaging. NIR photodetection is important for several applications, including optical communication, artificial vision, and health monitoring. Commercially available NIR photodetectors use inorganic materials such as InGaAs and HgCdTe, which are toxic, inflexible, and have limited tunability of their spectral response range. Conjugated polymers offer an alternative to these materials as they are considerably less toxic, flexible, and their spectral response range is tunable through molecular design. Thieno[3,4-b]pyrazine (TP) homopolymers show potential for NIR photodetectors due to their ability to absorb NIR light. However, TP homopolymers generally exhibit low solubility, which limits their application to devices. To improve solubility, TP homopolymers were functionalized with branched side chains, resulting in soluble materials with bandgaps as low as 0.64 eV. In addition, NIR photodetectors made from these materials exhibit specific detectivity values that are competitive with some of the top-performing polymers currently used in NIR photodetectors. Moreover, TP homopolymers are of relatively low synthetic complexity compared to current state-of-the-art conjugated polymers. The overall design, synthesis, characterization, and device data for these materials will be presented. Both absorption and emission are crucial for bioimaging, unlike NIR photodetection which only requires absorption. Fluorescence imaging allows for fast feedback and high sensitivity while being relatively inexpensive compared to traditional imaging methods. The NIR-I and NIR-II windows (700–900 and 1000–1700 nm, respectively) are ideal for fluorescence imaging due to the reduced absorption, autofluorescence, and scattering in these regions. Organic small molecule fluorophores have gained interest due to their low toxicity, fast excretion rates, tunability, and good biocompatibility. The overall design, synthesis, and characterization of several small molecule emitters will be presented.
Influence of Active Site Ligands and Nanoparticle Surfaces on Human Carbonic Anhydrase Isozymes
(North Dakota State University, 2010) Manokaran, Sumathra
Carbonic anhydrase (CA) is an ubiquitously distributed zinc containing metallo enzyme that catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Existence of 16 isoenzymes of CA in the animal kingdom has been known so far with varying subcellular and tissue distributions. Due to their involvement in many physiological and pathological processes, these isozymes have been the target for drug designing for the past 6 decades. The present study was designed with the aim of understanding the effect of active site ligands and nanoparticle surfaces on human carbonic anhydrase isozymes. In an effort to identify a fluorescent probe for carbonic anhydrases, the quantum yields and binding affinities of a variety of naphthalenesulfonamide derivatives with human carbonic anhydrase isozymes (hCAs) were determined. In this pursuit, a highly sensitive fluorescent probe, JB2-48 was identified. Experiments involving the above flurophore with hCA I unraveled the contributions of the sulfonamide moiety and the hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme-ligand complex. The fluorescence data revealed that the deprotonation of the sulfonamide moiety of the enzyme-bound ligand increases the fluorescence emission intensity as well as the lifetime of the ligand. This is manifested via the electrostatic interaction between the active site resident Zn2 + cofactor and the negatively charged sulfonamide group of the ligand. Evidence was provided that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. Investigations on the binding of the sulfonamide inhibitor, benzene sulfonamide (BS), with hCA isozymes II and VII, revealed that the binding is stabilized by polar interactions in the former case and hydrophobic interactions in the latter case. In addition, it was found that the binding of BS with hCA II is enthalpically driven at low temperatures, whereas it is entropically driven for its binding with hCA VII. Due to the prevalence of bipolar distribution of charges on hCA XII, the effects of the interaction of differently charged quantum dots, liposomes and polylysine on hCA XII were investigated. These charged particles were found to differently modulate the active site of the enzyme. The data revealed that whereas poly lysine and liposomes exhibited no influence on the binding and catalytic features of the enzyme, quantum dots had significant influence on the above features. Arguments were presented that the above differential feature exhibited by quantum dots, liposomes and poly lysine is encoded in the rigidity versus flexibility of the charged molecules. Studies on the denaturation of hCA isozymes II and XII unraveled their unfolding mechanism. It was found that the unfolding ofhCA XII followed a simple two state model from native to unfolded state; however hCA II unfolded with the formation of a stable intermediate.
Differential Modulation of the Structural and Functional Characteristics of Human Matrix Metalloproteinase Isozymes upon Binding to Different Ligands
(North Dakota State University, 2010) Ganguly, Bratati
Matrix metalloproteinases (MMPs) are a family of Zn2 + -dependent, Ca2 + -containing endoproteinases involved in tissue remodeling and degradation of the extracellular matrix (ECM). Human MMP isozymes are known to be involved in the progression and metastasis of many diseases like cancer, Alzheimer's, and etc. The different nanoparticles (e.g. gold nanoparticles, liposomes, and charged quantum dots) used in this study provides insights into nanoparticle-induced differential modulation in the structural-functional characteristics of MMP 7, 9 and 10 for better therapeutic intervention. To demonstrate the relationship between the rigid and flexible surfaces on the differential modulation of functional and structural characteristics of MMP-7, polylysine (PLL) and cationic gold nanoparticles (Au-CNP) were selected as representative examples. These cationic nano-structures were expected to serve as "soft" (flexible) and ''rigid" (hard) ligands, respectively. Steady-state kinetic analysis demonstrated that PLL induces activation and inhibition of MMP-7 at stoichiometric and super-stoichiometric concentrations respectively. Circular Dichroism spectroscopy was used to confirm that binding of Au-CNP to MMP-7 induces denaturation of the protein. In pursuit of understanding the molecular origin of the intrinsic selectivity in binding of human MMP isozymes to differently charged lipid membranes, steady-state kinetic studies and intrinsic tryptophan quenching studies were carried out. Results demonstrated that differently charged lipid membranes bind to all three MMPs; phosphotidylserine (POPS) liposomes are selective for MMP-7. The bipolar distribution of negative and positive charges on the surface of this enzyme dictates the binding of liposomes and perturbation of catalytic activity. An attempt to explain the molecular rationale for alternative binding modes of differently charged quantum dots (QDs) to the three MMPs, steady-state tryptophan quenching, steady-state kinetics, and time-resolved fluorescence measurements were carried out. Differently charged QDs bind to all the three MMP isozymes. Enzyme activity of these MMPs was perturbed upon binding to cationic and anionic QDs. Binding of MMPs to the differently charged QDs is reversible and is mediated via electrostatic interactions. Analysis of time-resolved fluorescence data indicates that the protein expenences different micro-environments, due to different distribution of intrinsic tryptophan residues (buried and exposed) on MMP isozymes or the existence of two distinct conformations of the protein. Binding to charged QDs perturbs enzyme activity of MMPs either by restricting the access of the substrate to the active-site or through allosteric modulation. In order to develop new isozyme-selective inhibitors, small molecule inhibitors (SMis) were designed, synthesized and screened for MMP-7, 9 and 10. Results indicate that hydroxamates and carboxylates are preferred SMis. Binding preference is based on either the micro-environments of the active-site pockets.
Mechanistic Investigation of Catalytic Chlorite Decomposition by Chlorite Dismutase
(North Dakota State University, 2021) Geeraerts, Zachary
Water purification processes often involve chlorine-based disinfectants which consequently leads to formation of chloro oxyanions (ClOx−), potent water-soluble oxidizing agents. These chlorine species are kinetically stable in water which allows them to persist and buildup to hazardous concentrations. Chlorite (ClO2−), a common contaminant in water purification processes, has been labeled as a top ten major water contaminant by the United States Environmental Protection Agency. Therefore, there is pressing interest in developing methods for removal of ClOx− species from water. Chlorite dismutases are heme b dependent enzymes that catalyze the unimolecular decomposition of ClO2− into chloride (Cl−) and molecular oxygen (O2) with remarkable efficiency. Catalytic O−O bond formation is a rare process in Nature as the only other well characterized example occurs in photosystems II. Clds are great candidates for bioremediation purposes and an excellent model for investigating catalytic O−O bond formation as the catalyzed reaction is not hindered by the need to pump protons. The goal of this work is to gain mechanistic insight into catalytic ClO2− decomposition by Cld and to investigate the structural features that tune the reaction pathway for productive O2 evolution. Cld from Klebsiella pneumoniae and Dechloromonas aromatica are the representatives used in this work. Since heme is the cofactor in these enzymes, a variety of spectroscopic tools have been utilized to probe the electrostatic landscape of the active site. Vibrational (resonance Raman and infrared), optical absorbance, and electron paramagnetic resonance spectroscopies were used to characterize the heme environment. Various ligand complexes were prepared to probe non-reactionary states of the enzymes while reactionary states were directly observed through use of stopped-flow spectrophotometry and freeze-quenched methods. Site directed mutagenesis studies of key amino acids were performed in combination with the above techniques to elucidate how changes in the electronics of the heme pocket alter catalytic activity. These studies have allowed for development of a proposed mechanism that describes the sequential steps and identification of the reactive intermediates leading to catalytic O2 production that accounts for the pH dependency of the reaction that was previously not understood.
Synthesis of Novel Biomass-derived Diol and Epoxide Monomers for Coatings Applications
(North Dakota State University, 2022) Sutton, Catherine
There are few monomers or precursors in the polymer industry that are more ubiquitous than bisphenol A (BPA). According to the CDC, about 5-6 billion pounds of BPA are produced worldwide annually. For the synthesis of coatings and polymeric materials, BPA is polymerized directly as a diol into polycarbonates or, to a lesser extent, glycidated into an epoxy monomer or resin. For coatings applications, BPA epoxy resins are utilized in protecting metal cans from acidic foods and beverages to heavy machinery like farm equipment from weather-related corrosion. In part, this popularity has led to scrutiny of the popular monomer from a few different perspectives. Since BPA is petroleum-derived, there is an effort to find a renewably sourced alternative from a sustainability perspective. Additionally, the structural similarity of BPA and the hormone estradiol make BPA an endocrine disruptor. This combined with its widespread applications, means BPA could be a larger issue than previously understood. To meet that challenge, many researchers, Sibi group included, have turned to biomass-derived “building-block” chemicals. Biomass feedstocks contain unique structures not easily obtained from petroleum sources. While avoiding detrimental structure-activity relationships associated with BPA, the newly synthesized compound would need to retain or mimic the structure-property relationships of BPA-containing polymers for coatings applications. The cellulosic monomer, 2,5-furandicarboxylic acid (FDCA), with exciting similarities to and some improvements upon petroleum-derived terephthalic acid was known, and its oxidative family of furans was being explored at the start of this project. A collection of furan diols was synthesized from 5-hydroxymethylfurfural (HMF), 2, 5-diformylfuran (DFF) and FDCA were synthesized by alkylation with various alkyl groups resulting in mono-, di-, and tetraalkylated diols, respectively. Depending on the alkyl group, certain materials properties were anticipated from these furanic diols. The diols were screened for estrogenic, androgenic, anti-thyroid activity via CALUX assays. The cytotoxicity of the diols were also determined via cell death studies. From those results, few low molecular weight furan diols do not exhibit any observable activity as endocrine disruptors or an observable cytotoxicity. Subsequently, a selection of furanic diols were glycidated for use in epoxy coating synthesis.
Examining Thieno[3,4-b]pyrazine Through a Multifaceted Lens: From Extended Ring Functionalization to Ambipolar-Acceptor Copolymers
(North Dakota State University, 2022) Culver, Evan
A class of materials known as conjugated polymers (CPs) has been shown to integrate the physical properties of organic plastics such as low-weight, flexibility, and synthetic modularity with electronic semiconducting properties typically found in inorganic materials. While a variety of parameters determine the resultant material’s conductivity, a crucial factor is the bandgap (Eg). Specifically, thieno[3,4-b]pyrazine (TP) has found success in generating low Eg CPs (i.e. Eg < 1.5 eV), largely in part due to its ambipolar identity. Two strategies to achieve Eg values < 1 eV include extending the conjugation of TP through ring fusion and pairing TP with strong electron accepting moieties. The investigation into extended ring TPs as low bandgap homopolymers was initially pursued with the synthesis of poly(acenaphtho[1,2-b]thieno[3,4-e]pyrazine), a record setting low Eg homopolymer. Upon this realization of driving Eg¬ values down through ring fusion on the pyrazine portion of TP, additional analogues were considered with 2λ4δ2-dithieno[3,4-b:3’,4’-e]pyrazine as one of the most promising candidates due to its predicted Eg of 0.14 eV. Efforts into this research have produced a variety of precursors and analogues, adding to the family of TPs for further study. A second strategy for Eg reduction is through the pairing of electronically mismatched units known as donors and acceptors. While this does reduce Eg, the underlying principles of the cause is disputed. Thus, to further understand the interactions in these types of copolymer systems, a small molecule study was designed with a strong donor, a strong acceptor, and the ambipolar unit TP in which six possible dimer configurations were synthesized and analyzed to determine the extent of donor-acceptor interactions. Lastly, an investigation into TP-acceptor alternating copolymers was carried out by pairing TP with two acceptors of varying accepting strength and contributions to polymer solubility. Because of the atypical design of these copolymers, a relatively new cross-coupling method known as direct arylation polymerization was used in their synthesis. The optimization of which produced a CP with an Eg of 0.93 eV. These results thus provide evidence for a new design motif for low bandgap CPs that further refines our understanding of donor-acceptor relationships.
New Insights into Apoptosis-Inducing Factor Mediated Pro-Survival Activity
(North Dakota State University, 2022) Birua, Sujata
Cancer is a group of diseases characterized by the uncontrolled growth of cells and is caused by the accumulation of genetic mutations that contribute to cell division, cell growth, and the DNA repair system. According to the American Cancer Society, more than 1.9 million new cancer cases will be diagnosed in 2022, hence there is a need to study new molecular mechanisms leading to tumorigenesis and develop novel treatment options.While significant research has been done to understand the underlying mechanisms, cancer still poses challenges as it 1) resists cell death, 2) activates metastasis, 3) sustains proliferative signaling, and 4) deregulates cellular metabolism. The present work explores the cancer-supporting role of apoptosis-inducing factor (AIF), a mitochondrial flavoprotein positioned at the convergence of the four hallmarks of cancer. AIF was initially characterized as an effector of caspase-independent death; however, increasing evidence has identified the physiological role of AIF in a variety of cancer, including colorectal, prostate, and pancreatic cancer. Expanding AIF activity studies in additional cancers, such as breast cancer, revealed the capability of AIF to modulate the consumption of biochemical substrates other than glucose, thus deregulating cellular metabolism. These alterations suggest the role of AIF in controlling a switch from metabolic flexibility to metabolic dependency, which can be exploited for therapeutic interventions. Moreover, AIF might be involved in mitochondrial biogenesis of breast cancer, unreported in cancer tested so far. In addition to their metabolic function, AIF serves as a signaling molecule that promotes cadherin switching in a 3-dimensional cell culture model of pancreatic ductal adenocarcinoma, which is associated with tumor growth. Finally. When grown in 3-dimensional culture conditions, pancreatic cancer cells were sensitized to a metabolic inhibitor in an AIF-dependent manner. Altogether, these AIF activities are functionally dissociable and demonstrate that AIF-mediated therapy has promise as a next-generations cancer treatment strategy.
Scrutinizing the Role of Ambipolar Units in the Donor-Acceptor Framework
(North Dakota State University, 2021) Anderson, Trent Eugene
Conjugated polymers (CPs) are a class of materials that contradict what is commonly considered when people hear the terms plastics and electronics. While these two phrases are generally considered exclusive, CPs combine the optical and electronic properties of inorganic materials and the flexibility and processability of traditional organic polymers and plastics. Research into CPs has resulted in an improved understanding of these compounds, leading to its application in the form of organic photovoltaics, organic light-emitting diodes, sensors, electrochromics, and field effects transistors. During this time, a number of models were developed for designing these polymers with desired characteristics, with the donor-acceptor framework becoming the most widely used model. This model utilizes electron-rich donor units and electron-deficient acceptor units that generate a material with a reduced energy difference between its frontier orbitals. Thieno[3,4-b]pyrazine (TP) based compounds are compounds used by the Rasmussen group and have distinct characteristics that has deemed it necessary to give it a new classification of an ambipolar unit within the donor-acceptor framework. TP has been previously classified as an acceptor unit within the donor-acceptor framework, but it has been shown to behave as both an acceptor and a donor simultaneously. In an effort to understand how the ambipolar unit behaves when paired with donor and acceptor units, a family of dimers was generated to determine the role of the ambipolar unit. Based on the findings from the dimer family, polymers of an alternating TP unit and different acceptors were generated to form a new family of acceptor-ambipolar polymers that also have desirable electronic characteristics with respect to the band gap and energy levels. This work provides a new insight on evaluating monomer units within the donor-acceptor framework as well as establishing a viable alternative for polymer design using ambipolar units.
Surface and Interface Effects on the Photoexcited Process of Silver Nanoclusters, and Lead & Cadmium Chalcogenide Nanocrystals
(North Dakota State University, 2020) Jabed, Mohammed Abu
The surface and interface of the metal nanoclusters and semiconducting nanomaterials play a key role in determining the electronic structure and overall photophysical properties. A single strand DNA stabilizes the metal nanoclusters, but it also influences the structural change, solvation free energy, and photophysical properties. On the other hand, surface and interface states in Pb and Cd chalcogenide nanomaterials affect the phonon mediated hot carrier relaxation. We applied DFT and DFT based non-adiabatic dynamics methods to study the surface and interface’s effects on the photoexcited processes. In the first part, we have studied the Ag nanoclusters' photophysical properties that are affected by the structural isomers, redox potential, nucleobase passivation, and cluster size. Ag nanoclusters are shown alternative reduction potential, which makes nanoclusters of singlet spin multiplicity thermodynamically favorable. Besides, the optically bright transition in the range of 2.5-3.5 eV is shown metal to ligand charge transfer. It is modulated by the s+p+d orbital mixing in the hole and electron states. We also simulate the charge transfer from the photoexcited PbS QD to organic dye (PDI) attached to the QD surface. Depending on the linker group and the dipole moment of neighboring passivating ligands, the PDI-QD conformations are varies. In response to structural change, the total dipole moment is modulated, changing its electronic structure and hence the photoexcited electron transfer rate from the PbS QD to PDI. We also investigate the inorganic-inorganic interactions in the PbCl2 bridged PbSe NPL and PbSe|CdSe Janus heterostructure. The energy dissipation rate of hot electrons is slower in NPL than the hot hole, while hot e-h relaxed to the band-edge by ≈1.0ps in the QD. The slower relaxation rate is rationalized by a large average intraband energy difference and smaller coupling term. Besides, the hot carriers in the NPL are spatially separated by ≈1.00 ps, which is a favorable condition for the carrier multiplication process. In Janus QD, (100) interfacial layer creates a structural mismatch in the CdSe part. Besides, the energy offset between the valance localized on PbSe and CdSe part is minimum in the PbSe Janus QD of an interface of (111) facet.
Designing Ru Catalysts for Selective C-H Bond Oxidation Reactions
(North Dakota State University, 2020) Herath, Hashini Nuradha Kumari
Typically, C-H bond oxidation proceeds via formation of high-valent metal oxo species. Attaining a high oxidation state of the metal complex is critical as it is the key step for many catalytic processes. Understanding ligand effects on structural and electronic changes of the metal complexes towards designing more robust catalysts is an effort of this dissertation. It will highlight initial attempts at developing novel ruthenium (Ru) catalysts and an analysis of their structural, electrochemical and spectroscopic properties. The catalytic behavior of the resulting complexes towards C-H bond hydroxylation reaction will also be shown. Chapter I introduces the background of C-H bond activation and hydroxylation reactions by Ru catalysts. Also, this chapter details the study of C-H bond hydroxylation mechanisms, reaction intermediates, the importance of C-H bond hydroxylation, electronic effects of ligands, and the pioneering work in this field. Chapter II describes development a of new Ru complex containing the pyridine alkoxide ligand, of general formula [Ru(tpy)(pyalk)Cl] (tpy = 2,2’:6’2”-terpyridine, pyalk = 2-(2′-pyridyl)-2-propanol). This chapter outlines the detailed synthesis, structural, electrochemical and spectroscopic properties of the complex by electrochemical techniques, UV Visible spectroscopy, NMR, mass spectrometry and X-ray crystallography. In order to overcome some limitations on project 1, new ruthenium complexes [Ru(MepyPO3H)(tpy)Cl] (1, tpy = 2,2’:6’2”-terpyridine , MepyPO3H = (pyridine-2-ylmethyl)phosphonic acid) and [Ru(bpyPO3H)(bpy)Cl] (2, bpy(PO3H2) = 2,2-bipyridine-6-phosphonic acid, bpy = 2,2-bipyridine) bearing phosphonate ligands were prepared and fully characterized. Catalytic properties of the complexes have been evaluated by testing their ability to catalyze C-H bond oxidation using a variety of sacrificial oxidants.
Investigating the Role of BECN1 Conformationally Flexible Regions and Invariant Cys-x-x-Cys Motifs in Autophagy
(North Dakota State University, 2020) Mukhopadhyay, Shreya
Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1, a key autophagy protein involved in autophagosome nucleation, comprises of a large, poorly conserved, N-terminal intrinsically disordered region (IDR); a flexible helical domain; a coiled-coil domain (CCD) that forms anti-parallel homodimers in the absence of other interacting partners; and a β-α repeated autophagy specific domain (BARAD). The IDR of higher eukaryotes includes a BCL2 homology 3 domain (BH3D), which undergoes dramatic disorder-to-helix transitions upon binding to anti-apoptotic and anti-autophagic BCL2s. We show that the BH3D is not required for starvation-induced autophagy upregulation suggesting that BCL2-binding to the BH3D does not directly impede a pro-autophagy function of the BH3D, rather it may impact structure, oligomerization, interactions and function of other BECN1 domains. CCD C-terminal residues, named the overlap helix (OH), pack in two mutually-exclusive states stabilized by the same interface residues: against either the partner helix in a CCD dimer or the BARAD. We show that mutation of these interface residues abrogates starvation-induced autophagy upregulation. Together with our complementary structural studies, this suggests that autophagy-inactive BECN1 adopts conformations preventing the BARAD from membrane-association, with BECN1 heterodimerization with ATG14 or UVRAG disrupting this inhibitory conformation. In the BECN1 homodimer, the OH packs against a nuclear export signal sequence (NES) at the N-terminus of the partner CCD. We show that when released from this interaction, the NES can interact with the complex of the nuclear exporter, Chromosomal Region Maintenance 1 protein and a GTP-bound small G-protein, Ran. This interaction is essential for BECN1 export to the cytoplasm, and for autophagy. Two invariant CxxC motifs bookend the IDR. We find that both CxxC motifs are required, but the intervening IDR is less important, for starvation-triggered upregulation of autophagy. We demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio. Further, mutation of the invariant cysteines or treatment with reducing agent abrogates Zn2+ co-ordination, demonstrating that the invariant CxxC motifs are responsible for binding Zn2+. We use diverse biophysical methods to show that Zn2+-binding impacts the conformation and structural transitions of the BECN1 IDR, thereby playing an important role in regulating autophagy.
Enzyme Behavior in Synthetic Materials and Structural Implications for Rational Design
(North Dakota State University, 2020) Farmakes, Jasmin Kaye
Combining enzymes with synthetic materials is the new frontier of biocatalysis, materials science, and protein engineering. Enzymes are biological macromolecule catalysts with incredible efficiency and specificity that are desirable for use in a variety of different fields. However, commercial applications have been limited by the stability and reusability of un-altered enzymes. An avenue for overcoming the challenges to harnessing enzyme power is to combine enzymes with materials to create an enzymatically-active material that has enhanced stability and activity. Unfortunately, the catalytic activity of the hybrid material is often lower than that of the enzyme alone. The activity of an enzyme is directly dependent on its structure and dynamics. Therefore, a deeper understanding of enzyme structure and dynamics upon incorporation into materials will provide the data necessary to rationally design enzymatically-active materials with the desired features. This dissertation explores the behavior of a model enzyme, T4 Lysozyme, with two different artificial material systems, metal-organic frameworks and polyethylene glycol. The underlying structural rationale for the behavior is probed using a variety of techniques, notably, Electron Spin Paramagnetic Resonance. Herein, the implications of structural alterations on activity and opportunities for exploitation are discussed. T4 Lysozyme is a perfect model for this study because it has a well characterized structure-activity relationship, thus providing a vast literature understanding which can be pulled from to verify and assist with interpretation of data. The structural basis of enzyme activity alteration in artificial materials can be used to rationally design systems with desired characteristics. After successfully demonstrating the tunability of proteins in artificial materials using T4L as a model, human Cu/Zn superoxide dismutase 1 was chosen for continuing studies due to its importance in diseased states. However, the superoxide dismutase mutant chosen is aggregation prone, which makes it difficult to express recombinantly in large amounts. Therefore, an efficient protocol for producing the superoxide dismutase protein was developed to set the stage for future studies.
Seeing the Forest for the Trees: An Exploration of Student Problem Solving and Reasoning with 1H NMR Spectral Features
(North Dakota State University, 2020) Anderson, Shannon Yun
Nuclear magnetic resonance (NMR) spectroscopy is vital to synthesis and provides rich problem-solving opportunities for organic chemistry students. However, little is known about 1H NMR spectroscopy instruction or how students use spectral features in solving. The goal of this dissertation research was to examine how students learn about and solve 1H NMR spectroscopy problems. Organic chemistry textbooks were analyzed for the ways in which spectral features were introduced and incorporated into worked examples and practice problems. Spectral features like the number of signals and chemical shift were covered by problems more frequently, while integration was covered least. Think-aloud interviews were completed to identify the operators students utilized in their problem-solving processes, and extra credit problem sets were designed and administered to students at three different universities to examine whether students could correctly perform each individual type of operator. While students could perform operators, it was unclear if students knew how and when to use the operators. To fill this knowledge gap, multiple choice assessment questions were developed and administered to students at three different large universities. Coding schemes were developed to identify and describe students’ use of task features and inferences, and regression analyses were completed to discern which areas of reasoning led to success in solving. A majority of students did not identify using any critical spectral features in written explanations. Regression analyses revealed that the inferences students made, and not the task features they paid attention to, were most significantly associated with success in structural predictions; a majority of students made solely correct inferences in their reasoning explanations. When a mixture of correct and incorrect inferences were made, a majority of those students were unable to answer the questions correctly. These findings suggest that students may know enough to solve simple 1H NMR spectroscopy problems, but may lack knowledge about specific spectral features which could impact overall solving success. Students may require considerable support in deciphering the critical features in 1H NMR spectroscopy problems and developing robust, correct inferences across all spectral features.
Influence of Organic and Inorganic Passivation on the Photophysics of Cadmium Chalcogenide and Lead Chalcogenide Quantum Dots
(North Dakota State University, 2020) Lystrom, Levi Aaron
Quantum dots (QDs) are promising materials for photovoltaic (PV) and light-emitting diode (LED) applications due to their unique properties: photostability, size-tunable absorptivity, and narrow line-width emission. These properties are tailored by surface passivations by ligands. However, ligands used in the synthesis of colloidal QDs need to be exchanged with ligands designed for specific applications. The mechanism behind ligand exchange is not well understood. Density functional theory (DFT) is utilized to gain fundamental understanding of ligand exchange (LE) and the resulting effect on the photophysics of QDs. Experimental studies show that phenyldithiocarbamates (PTCs) derivatives can improve the photocurrent of QD-based PVs. Our calculations show that the PTC undergoes decomposition on the CdSe QD surface. Decomposed products of PTCs strongly interact with the surface of QDs, which could cause unforeseen challenges during the implementation of these functionalized QDs in PVs. Secondly, we studied the mechanism of photoluminescence (PL) enhancement by hydride treatment. In experiments, the PL increases by 55 times, but the mechanism is unclear. We found that hydride can interact with surface Se2- producing H2Se gas and passivate surface Cd2+. These interactions result in optically active QDs. Thiol derivatives can also improve PL when LE results in low surface coverage of thiols. The PL is quenched if LE is performed at high concentrations and acidic environments. DFT simulations reveal three scenarios for the thiol interacts with QDs: coordination of thiol, networking between surface and/or other ligands, or thiolate formation. It is the last scenario that was found to be responsible for PL quenching. Lastly, PbS(e)/CdS(e) core/shell QDs are investigated to obtain relaxation rates of electron and hole cooling via interactions with phonons. The band structure of the core/shell QDs facilitates carrier multiplication (CM), a process that generates multiple charge carrier pairs per one absorbed photon. It is thought that CM is facilitated because there are interface associated states that reduce carrier cooling. Non-Adiabatic Molecular Dynamics (NAMD) simulations show that this hypothesis is correct and PbSe/CdSe carrier cooling is about two times slower compared to PbS/CdS due to weaker coupling to optical phonons.
New Strategies for Transition Metal Catalyzed C-C and C-N Bond Formation
(North Dakota State University, 2018) Kilaru, Praveen
Transition metal catalysis emerged as an essential tool in the field of organic chemistry. In this context, transition metal catalyzed C-H bond functionalization is considered as an alluring strategy as it occurs with the high atom-and-step economy. In the recent years, significant attention has been paid for the conversion of C-H bond into C-X (X = C, N, O, S, P..etc) bonds using transition metal catalysts. This thesis presents the development of new catalytic systems for the construction of C-C and C-N bonds through late transition metal-mediated C-H activation and decarboxylation reactions. Chapter 1 introduces the background of transition metal catalyzed C-H bond functionalization. This chapter provides reported catalytic methods for the conversion of arene C-H bonds into various functional groups through transition metal mediated chelation-assisted C-H bond activation. Chapter 2 describes the development of a new method for the synthesis of oxindoles via intramolecular alkene hydroarylation with N-aryl acrylamides using a Ru(II)/N-heterocyclic carbene (NHC) catalyst system. This reaction occurs with good substrate scope and synthetically useful tolerance of functional groups and does not require the assistance of additional directing group. Preliminary mechanistic results support a tandem sequence involving amide-directed aromatic C-H bond activation and intramolecular alkene arylmetalation. Chapter 3 describes ruthenium-based decarboxylative alkenylation of heteroarenes through carboxylate directed C-H bond functionalization. The decarboxylative functionalization of heteroarenes occurs with high regioselectivity and a broad range of functional group tolerance. This decarboxylation proceeds without stoichiometric amounts of bases or oxidants and it is applicable for functionalization of various heteroarenes such as indole, pyrrole, thiophene, benzothiophene, and benzofuran at both C-2 and C-3 positions. The current protocol provides a straightforward approach for the synthesis of trisubstituted olefins with heteroarenes. Chapter 4 explains the development of Rh/Ag-bimetallic catalyst system for decarboxylative amidation of ortho-substituted benzoic acids with 3-aryldioxazolones. The nature of ortho-substituents determines regioselectivity of this reaction through two forms of proposed chelation assistance: (1) A wide range of non-directing ortho-substituents led to ortho-amidation products via carboxylate-directed C-H amidation and subsequent decarboxylation. (2) 2-Pyridyl and analogous DGs led to ipso-amidation products via DG-assisted decarboxylation and subsequent amidation.
Synthesis and Design of Thiophene Materials: Effects of Ring Fusion and Metal Coordination
(North Dakota State University, 2019) Konkol, Kristine Louise
Conjugated organic materials comprise a field of materials chemistry focused on the development of semiconducting organic plastics, popular applications of which are plastic solar cells and display technologies. One of the reasons these materials have gained so much attention is that their optical and electronic properties can be tuned through engineering at the molecular level. Thiophene, an aromatic heterocycle, is a popular building block in the synthesis of many conjugated materials, prized for both the ease in which it can be synthetically functionalized and its ability to form highly conductive and low band gap materials. The Rasmussen group has previously reported on the generation of two classes of materials, the inorganic metal thiophenedithiolenes and the fused-ring heterocycle unit thieno[3,4-b]pyrazine (TP), both of which have applications in conducting materials. In an effort to expand upon the applicability and versatility of these materials, a series of interconnected projects were performed to further tune their optical, electronic, and physical (e.g. solubility) properties. This involved synthetic molecular design, including judicious consideration of structure-function relationships, and characterization of the resulting materials. Highlights include a sterics vs. electronics consideration of the catalyzed hydrodebromination of the molecular building-block 2,3,5-tribromothiophene, variation of the coordinating metal in thiophenedithiolenes to tune the optics and electronics, and characterization of the effects of ring-fusion on TP-based terthienyl homopolymers. Additionally, a new application of the TP monomer was found, whereby it was successfully incorporated as a bridging ligand into a multi-metallic Ru(II) supramolecular assembly, which demonstrated good metal-metal communication.
Structural Basis for the Regulation of a Conserved TonB-dependent Iron Transport System via Cell-surface Signaling in Gram-negative Bacteria
(North Dakota State University, 2017) Jensen, Jaime Lea
Cell-surface signaling (CSS) pathways are highly conserved systems in Gram-negative bacteria that allow the cell to efficiently respond to environmental stimuli through transcriptional regulation. Three distinct proteins are involved in this process: an outer membrane (OM) protein that senses the extracellular signal, an inner membrane (IM) sigma regulator protein that transmits the signal from the OM protein to the cytoplasm, and an extracytoplasmic function (ECF) sigma factor that initiates transcription of stimulus response genes. One such CSS pathway regulates bacterial iron acquisition- an essential process for bacterial survival and pathogenesis. Under iron-limited conditions, expression of the OM transporter is upregulated by signal transduction through the IM protein to the sigma factor. The goal of this work is to provide a structural rationale for distinctive signal transduction through the CSS pathway that regulates ferric siderophore uptake in Gram-negative bacteria, by structurally characterizing these proteins from Pseudomonas capeferrum, with a focus on the IM protein, PupR. The solution structures of an OM transporter, PupA, an OM transducer, PupB, a PupAPupB-NTSD chimera, and the OM proteins with the PupR C-terminal cell-surface signaling domain (CCSSD) were probed by SEC-SAXS to examine global architectural differences amongst the OM proteins. The X-ray crystal structure of the PupB N-terminal signaling domain (NTSD):PupR-CCSSD complex was determined. The PupB-NTSD exhibits a conserved βαβ-repeat motif. Unexpectedly, the CCSSD subdomain contains the same fold, which is the first time this fold had been identified at a protein’s C-terminus. The other subdomain of the CCSSD, designated the C-terminal juxtamembrane (CJM) subdomain, has a novel, β-solenoid-like motif. Analysis of the CCSSD by CD spectroscopy and SEC-SAXS indicated that the domain is highly flexible, and is significantly stabilized by the PupB-NTSD. Concurrently, the PupB-NTSD structure was determined by NMR, and contrasted with published NTSDs to evaluate structural variation that may account for disparities in functionality. The PupR N-terminal anti-sigma domain (ASD) was solved by X-ray crystallography and presents as a dimer in solution- the first description of a transmembrane ASD to assume an oligomeric form. Structural characterization of these proteins suggests novel implications for CSS through the TonB-dependent ferric siderophore uptake pathway.
Understanding the Interaction Between Enzymes and Nanomaterials
(North Dakota State University, 2019) Neupane, Sunanda
The rapid development of nanoparticles (NPs) has impacted many fields including energy efficiency, material science, biosensing, and medical therapeutics. Recently, NPs have been utilized to immobilize enzymes. The so-formed enzyme- NP complex show great potential to increase the reusability of enzymes and catalytic efficiencies. Enzyme-NP complex can also advance enzyme delivery for therapeutics where NPs serve as the enzyme carrier. In all applications, the contact of NPs with biomacromolecules, especially proteins, is either necessary or inevitable, which can lead to alterations in adsorbed enzyme structure and function. In biocatalysis, such changes often reduce the desired catalytic activity; in living organisms these changes can even cause protein malfunction, raising concerns about public health and nanotoxicity. Therefore, understanding the correlation of enzyme structure and activity upon contact with NPs is essential. While enzyme activities can often be determined, the details of enzyme structural changes caused by NPs are underexplored for most enzyme-NP complexes. Obtaining the structural information is challenging due to the relatively large size of the complexes, high heterogeneity in enzyme binding, and complexities caused by the presence of NPs which limit most structure determination approaches. These challenges were overcome using a set of biophysical techniques especially site-directed spin labeling (SDSL) with Electron Paramagnetic Resonance (EPR). SDSL-EPR can measure site-specific structural information in the native state of enzyme/NP systems, regardless of the complexity, primarily due to its “penetrating” power which is only sensitive to the motion of the spin label. The focus of this dissertation was on T4 lysozyme (T4L), a representing model enzyme proven useful in many works. Gold Nanoparticles (AuNPs), Gold Nanorods (AuNRs), Silica Nanoparticles (SiNPs), and Carbon Nanotubes (CNTs) were the studied NPs. The interaction of T4L with each NPs was unique. The local structural information and the orientation of T4L in each NP was revealed based on which the possible docking mechanism for each case was proposed. The ultimate goals to reveal the structure-function relationship of enzymes on NPs and utilize this information to fine-tune enzyme adsorption on various NPs to 1) avoid NPs aggregation and 2) optimize NPs as enzyme carriers were met.

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