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ABSTRACT Manipulation of biological targets using synthetic or naturally occurring organic compounds has been the focal point of medicinal chemistry. The work described herein centers on the synthesis of organic small molecules that are targeted either to cell surface receptors, to the ribosomal catalytic center or to human immunodeficiency virus reverse transcriptase. Bleomycins (BLMs) are a family of naturally occurring glycopeptidic antitumor agents with an inherent selectivity towards cancer cells. DeglycoBLM, which lacks the sugar moiety of bleomycin, has much lower cytotoxicity in cellular assays. A recent study using microbbuble conjugates of BLM and deglycoBLM showed that BLM was able to selectively bind to breast cancer cells, whereas the deglyco analogue was unable to target either the cancer or normal cells. This prompted us to further investigate the role of the carbohydrate moiety in bleomycin. Fluorescent conjugates of BLM, deglycoBLM and the BLM carbohydrate were studied for their ability to target cancer cells. Work presented here describes the synthesis of the fluorescent carbohydrate conjugate. Cell culture assays showed that the sugar moiety was able to selectively target various cancer cells. A second conjugate was prepared to study the importance of the C-3 carbamoyl group present on the mannose residue of the carbohydrate. Three additional fluorescent probes were prepared to improve the uptake of this carbohydrate moiety into cancer cells. Encouraged by the results from the fluorescence experiments, the sugar moiety was conjugated to a cytotoxic molecule to selectively deliver this drug into cancer cells. The nonsense codon suppression technique has enabled researchers to site specifically incorporate noncanonical amino acids into proteins. The amino acids successfully incorporated this way are mostly α-L-amino acids. The non-α-L-amino acids are not utilized as substrates by ribosome catalytic center. Hoping that mutations near the ribosome peptidyltransferase site might alleviate its bias towards α-L-amino acids, a library of modified ribosomes was generated. Analogues of the naturally occurring antibiotic puromycin were used to select promising candidates that would allow incorporation of non-α-L-amino acids into proteins. Syntheses of three different puromycin analogues are described here. The reverse transcriptase enzyme from HIV-1 (HIV-1 RT) has been a popular target of HIV therapeutic agents due to its crucial role in viral replication. The 4-chlorophenyl hydrazone of mesoxalic acid (CPHM) was identified in a screen designed to find inhibitors of strand transfer reactions catalyzed by HIV-1 RT. Our collaborators designed several analogues of CPHM with different substituents on the aromatic ring using molecular docking simulations. Work presented here describes the synthesis of eight different analogues of CPHM.
ContributorsPaul, Rakesh (Author) / Hecht, Sidney M. (Thesis advisor) / Moore, Ana L (Committee member) / Rose, Seth D (Committee member) / Arizona State University (Publisher)
Created2013
Description
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Dissolved inorganic carbon (DIC) and dissolved organic carbon (DOC) are crucial nutrients for autotrophic and heterotrophic microbial life, respectively, in hydrothermal systems. Biogeochemical processes that control amounts of DIC and DOC in Yellowstone hot springs can be investigated by measuring carbon abundances and respective isotopic values. A decade and a half of field work in 10 regions within Yellowstone National Park and subsequent geochemical lab analyses reveal that sulfate-dominant acidic regions have high DOC (Up to 57 ppm C) and lower DIC (up to 50 ppm C) compared to neutral-chloride regions with low DOC (< 2 ppm C) and higher DIC (up to 100 ppm C). Abundances and isotopic data suggest that sedimentary rock erosion by acidic hydrothermal fluids, fresh snow-derived meteoric water, and exogenous carbon input allowed by local topography may affect DOC levels. Evaluating the isotopic compositions of DIC and DOC in hydrothermal fluids gives insight on the geology and microbial life in the subsurface between different regions. DIC δ13C values range from -4‰ to +5‰ at pH 5-9 and from -10‰ to +3‰ at pH 2-5 with several springs lower than -10‰. DOC δ13C values parkwide range from -10‰ to -30‰. Within this range, neutral-chloride regions in the Lower Geyser Basin have lighter isotopes than sulfate-dominant acidic regions. In hot springs with elevated levels of DOC, the range only varies between -20‰ and -26‰ which may be caused by local exogenous organic matter runoff. Combining other geochemical measurements, such as differences in chloride and sulfate concentrations, demonstrates that some regions contain mixtures of multiple fluids moving through the complex hydrological system in the subsurface. The mixing of these fluids may account for increased levels of DOC in meteoric sulfate-dominant acidic regions. Ultimately, the foundational values of dissolved carbon and their isotopic composition is provided in a parkwide study, so results can be combined with future studies that apply different sequencing analyses to understand specific biogeochemical cycling and microbial communities that occur in individual hot springs.
ContributorsBarnes, Tanner (Author) / Shock, Everett (Thesis advisor) / Meyer-Dombard, D'Arcy (Committee member) / Hartnett, Hilairy (Committee member) / Arizona State University (Publisher)
Created2023
Description
Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer. An understanding of the variables which impact mutation rates and their estimation is necessary to place mutation rate estimates in their proper contexts. To better understand mutation rate estimates, this research investigates the impact of temperature upon transcription rate error estimates; the impact of growing cells in liquid culture vs. on agar plates; the impact of many in vitro variables upon the estimation of deoxyribonucleic acid (DNA) mutation rates from a single sample; and the mutational hazard induced by expressing clustered regularly interspaced short palindromic repeat (CRISPR) proteins in yeast. This research finds that many of the variables tested did not significantly alter the estimation of mutation rates, strengthening the claims of previous mutation rate estimates across the tree of life by diverse experimental approaches. However, it is clear that sonication is a mutagen of DNA, part of an effort which has reduced the sequencing error rate of circle-seq by over 1,000-fold. This research also demonstrates that growth in liquid culture modestly skews the mutation spectrum of MMR- Escherichia coli, though it does not significantly impact the overall mutation rate. Finally, this research demonstrates a modest mutational hazard of expressing Cas9 and similar CRISPR proteins in yeast cells at an un-targeted genomic locus, though it is possible the indel rate has been increased by an order of magnitude.
ContributorsBaehr, Stephan (Author) / Lynch, Michael (Thesis advisor) / Geiler-Samerotte, Kerry (Committee member) / Mangone, Marco (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2023
Description
In the realm of biosensors and nanotechnology, deoxyribonucleic acid (DNA) nanosensors have demonstrated tremendous potential across diverse real-world applications, from environmental monitoring to healthcare diagnostics. Fabrication of nanosensors allows assembling and designing of DNA molecules at nanoscale with high precision and versatility. Such fabricating DNA nanosensors are quite time consuming. Hence it is important to store them in batches. However synthetic DNA molecules can be prone to degradation over time, especially when exposed to various environmental factors like light, heat, or any other chemical contaminants. To address this issue, a shelf life study of DNA nanosensors using various lyoprotectant conditions was carried out to determine the long term stability of such sensors. This study involves fabrication of the dendritic, double - stranded DNA nanosensors involving five strands L1 through L5 conjugated with pHAb fluorophores via N-hydroxysuccinimide ester reaction and acetylcholinesterase (AChE) enzyme, a core component of the sensor. This sensor was originally a fluorescent ACh-selective nanosensors designed to accommodate the BTX ligand, AChE to image the ACh release in the submandibular region of the living mice to report real time quantitative endogenous ACh release triggered by electrical stimulation. AChE enzyme is a good receptor to detect acetylcholine release in the Peripheral Nervous System (PNS). The primary objective of the study was to assess DNA nanosensors with AChE, however due to the intricate interactions, non-specific binding and cost-effectiveness, the shelf life study was carried out separately. The shelf study includes observing DNA nanosensors with different disaccharide lyoprotectants like trehalose and sucrose that were analyzed under different temperature conditions: room temperature (25ºC) and at 50 ºC for different time intervals, over a week time. Also, Observing AChE with various protectants under 50 ºC with and without lyoprotectants for various time intervals like 24 hours and 48 hours. To replicate the real-world transit scenarios, the study also involves test-shipment of the samples with lyoprotectants for 2-3 days to both cross-country and local (in-state). As a result, the use of lyoprotectants, particularly trehalose, has proven to be more resilient and effective in preserving the stability and integrity of DNA nanosensors and Acetylcholinesterase (AChE) enzymes
ContributorsSrinivasan, Nikita (Author) / Clark, Heather A (Thesis advisor) / Ma, Kristine Y (Committee member) / Beeman, Scott (Committee member) / Arizona State University (Publisher)
Created2023
Description
Urease, an amidohydrolase, is an essential ingredient in the emerging engineering technique of biocementation. When free urease enzyme is used this carbonate precipitation process is often referred to as enzyme induced carbonate precipitation (EICP). To date, most engineering applications of EICP have used commercially available powdered urease. However, the high cost of commercially available urease is a major barrier to adoption of engineering applications of EICP in practice. The objective of this dissertation was to develop a simple and inexpensive enzyme production technique using agricultural resources. The specific objectives of this dissertation were (i) to develop a simple extraction process to obtain urease from common agricultural resources and identify a preferred agricultural resource for further study, (ii) to reduce the cost of enzyme production by eliminating the use of a buffer, centrifugation, and dehusking of the beans during the extraction process, (iii) investigate the stability of the extracted enzyme both in solution and after reduction to a powder by lyophilization (freeze-drying), and (iv) to study the kinetics of the extracted enzyme.
The results presented in this dissertation confirmed that inexpensive crude extracts of urease from agricultural products, including jack beans, soybeans, and watermelon seeds, are effective at catalyzing urea hydrolysis for carbonate precipitation. Based upon unit yield, jack beans were identified as the preferred agricultural resource for urease extraction. Results also showed that the jack bean extract retained its activity even after replacing the buffer with tap water and eliminating acetone fractionation, centrifugation, and dehusking. It was also found that the lyophilized crude extract maintained its activity during storage for at least one year and more effectively than either the crude extract solution or rehydrated commercial urease. The kinetics of the extracted enzyme was studied to gain greater insight into the optimum concentration of urea in engineering applications of EICP. Results showed higher values for the half-saturation coefficient of the crude extract compared to the commercial enzymes.
The results presented in this dissertation demonstrate the potential for a significant reduction in the cost of applying EICP in engineering practice by mass production of urease enzyme via a simple extraction process.
ContributorsJavadi, Neda (Author) / Kavazanjian, Edward (Thesis advisor) / Khodadadi Tirkolaei, Hamed (Committee member) / Hamadan, Naser (Committee member) / Delgado, Anca (Committee member) / Arizona State University (Publisher)
Created2021
Description
The complex network of the immune system defends the human body against infection, providing protection from pathogens. This work aims to improve preparation and structural knowledge of two proteins on opposite sides of the immune system spectrum. The first protein, secreted autotransporter toxin (Sat) is a class I serine protease autotransporter of Enterobacteriaceae (SPATE) that has cytotoxic and immunomodulatory effects on the host. Previous studies on Sat show its ability to aid in bacterial colonization and evasion of the immune system. This work improves the stability of Sat by making mutations to the active serine protease motif (GDSGS) while inhibiting remaining activity with reversible and irreversible serine protease inhibitors. Characterization of Sat by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography led to the first structural studies of Sat by x-ray crystallography and cryo-EM. Human leukocyte antigen class I proteins play an important role in the adaptive immune system by presenting endogenous viral peptides at the cell surface for CD8+ T cell recognition. In vitro production of HLA-I proteins is a difficult task without endoplasmic reticulum chaperones as present in vivo. Disulfide bond formation, folded light chain and a peptide bound are all key to refolding the HLA-I heavy chain for complex formation. The work presented in this dissertation represents systematic studies aimed at improving the production of HLA-I proteins in vitro in bacterial expression systems. Optimization of every step of the preparation was investigated providing higher expression yields, quality of inclusion bodies, and refolding improvements. With further improvements in the future, this work forms the basis for a more efficient small and large-scale production of HLA-I molecules for functional and structural studies.
ContributorsKiefer, Dalton (Author) / Anderson, Karen (Thesis advisor) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2024
Description
The thylakoid membranes of oxygenic photosynthetic organisms contain four large membrane complexes vital for photosynthesis: photosystem II and photosystem I (PSII and PSI, respectively), the cytochrome b6f complex and ATP synthase. Two of these complexes, PSII and PSI, utilize solar energy to carry out the primary reaction of photosynthesis, light induced charge separation. In vivo, both photosystems associate with multiple antennae to increase their light absorption cross section. The antennae, Iron Stress Induced A (IsiA), is expressed in cyanobacteria as part of general stress response and forms a ring system around PSI. IsiA is a member of a large and relatively unexplored antennae family prevalent in cyanobacteria. The structure of the PSI-IsiA super-complex from the cyanobacteria Synechocystis sp. PCC 6803 was resolved to high resolution, revealing how IsiA interacts with PSI as well as the chlorophyll organization within this antennae system. Despite these structural insights, the basis for the binding between 18 IsiA subits and PSI is not fully resolved. Several IsiA mutants were constructed using insights from the atomic structure of PSI-IsiA, revealing the role of the C-terminus of IsiA in its interaction with PSI.
ContributorsLi, Jin (Author) / Mazor, Yuval (Thesis advisor) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2024