ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Displaying 1 - 10 of 13
Filtering by
- All Subjects: Biochemistry
Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
ContributorsLuu, Dustin Dean (Author) / Van Horn, Wade D (Thesis advisor) / Redding, Kevin E (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
ContributorsJernigan, Rebecca Jeanne (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2022
Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic.
Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane.
Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021
Description
Osteocalcin (Oc) is the most abundant non-collagen protein found in the bone, but its precise function is still not completely understood. Three glutamic acid (Glu) residues within its sequence are sites for vitamin K-dependent post-translational modification, replacing a hydrogen with a carboxylate located at the γ-carbon position, converting these to γ-carboxyglutamic acid (Gla) residues. This modification confers increased binding of Oc to Ca2+ and hydroxyapatite matrix. Presented here, novel metal binding partners Mn2+, Fe3+, and Cr3+ of human Oc were determined, while the previously identified binders to (generally) non-human Oc, Ca2+, Mg2+, Pb2+ and Al3+ were validated as binders to human Oc by direct infusion mass spectrometry with all metals binding with higher affinity to the post-translationally modified form (Gla-Oc) compared to the unmodified form (Glu-Oc). Oc was also found to form pentamer (Gla-Oc) and pentamer and tetramer (Glu-Oc) homomeric self-assemblies in the absence of NaCl, which disassembled to monomers in the presence of near physiological Na+ concentrations. Additionally, Oc was found to form filamentous structures in vitro by negative stain TEM in the presence of increased Ca2+ titrations in a Gla- and pH-dependent manner. Finally, by combining circular dichroism spectroscopy to determine the fraction of Gla-Oc bound, and inductively-coupled plasma mass spectrometry to quantify total Al concentrations, the data were fit to a single-site binding model and the equilibrium dissociation constant for Al3+ binding to human Gla-Oc was determined (Kd = 1.0 ± 0.12 nM). Including citrate, a known competitive binder of Al3+, maintained Al in solution and enabled calculation of free Al3+ concentrations using a Matlab script to solve the complex set of linear equations. To further improve Al solubility limits, the pH of the system was lowered to 4.5, the pH during bone resorption. Complementary binding experiments with Glu-Oc were not possible due to the observed precipitation of Glu-Oc at pH 4.5, although qualitatively if Glu-Oc binds Al3+, it is with much lower affinity compared to Gla-Oc. Taken together, the results presented here further support the importance of post-translational modification, and thus adequate nutritional intake of vitamin K, on the binding and self-assembly properties of human Oc.
ContributorsThibert, Stephanie (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the
three-dimensional structure of protein targets has the potential to accelerate the process
for screening drug candidates. X-ray crystallography has proven to be the most used and
indispensable technology in structural-based drug discovery. The provided
comprehensive structural information about the interaction between the disease-related
protein target and ligand can guide the chemical modification on the ligand to improve
potency and selectivity. X-ray crystallography has been upgraded from traditional
synchrotron to the third generation, which enabled the surge of the structural
determination of macromolecular. The introduction of X-ray free electron laser further
alleviated the uncertain and time-consuming crystal size optimization process and
extenuated the radiation damage by “diffraction before destruction”. EV-D68 2A
protease was proved to be an important pharmaceutical target for acute flaccid myelitis.
This thesis reports the first atomic structure of the EV-D68 2A protease and the structuresof its two mutants, revealing it adopting N-terminal four-stranded sheets and C-terminal
six-stranded ß-barrels structure, with a tightly bound zinc atom. These structures will
guide the chemical modification on its inhibitor, Telaprevir. Integrin ⍺Mβ2 is an integrin
with the α I-domain, related to many immunological functions including cell
extravasation, phagocytosis, and immune synapse formation, so studying the molecular
ligand-binding mechanism and activation mechanism of ⍺Mβ2 is of importance. This
thesis uncovers the preliminary crystallization condition of ⍺Mβ2-I domain in complex
with its ligand Pleiotrophin and the initial structural model. The structural model shows consistency with the previous hypothesis that the primary binding sites are metal iondependent
adhesion sites on ⍺Mβ2-I domain and the thrombospondin type-1 repeat (TSR)
domains of Pleiotrophin. Drug molecules with high potency and selectivity can be
designed based on the reported structures of the EV-D68 2A protease and ⍺Mβ2-I domain
in the future.
ContributorsLiu, Chang (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Purification, Characterization, and Structural Determination of Proteins Vital to Infectious Disease
Description
The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that used small angle X-ray scattering (SAXS) in combination with computation analysis to generate a SAXS-refined structure. The SAXS-refined structure closely resembled the NMR structure (PDB: 2MU4) which contains a hydrophobic cavity inside the protein that could be used for drug discovery purposes. The full-length domain of Flpp3 purified from the outer membrane of E. coli was also studied with a combination of biophysical characterization methods. Mass spectrometry and western blot analysis confirmed Flpp3 being translocated to the outer membrane, while SDS-PAGE confirmed the purity of Flpp3 in the monomeric form after size exclusion chromatography. Using Circular Dichroism (CD) the monomeric form of Flpp3 was shown to be almost fully refolded into having a primarily β-stranded secondary structure. This information advances the progress of both tularemia research and outer membrane protein research as no natively folded outer membrane protein structures have been solved for F. tularensis.The second protein worked on in this dissertation is the nonstructural protein 15 from SARS-CoV-2, also called NendoU. Nsp15 is an endoribonuclease associated with aiding the virus responsible for the current COVID-19 pandemic in evasion of the immune system. An inactive mutant of Nsp15 was studied with both negative stain electron microscopy and cryogenic electron microscopy (Cryo-EM) in the presence of RNA or without RNA present. The initial findings of negative stain electron microscopy of Nsp15 with and without RNA showed a difference in appearance. Negative stain analysis of Nsp15 is in the presence of a 5nt RNA sequence in low salt conditions shows a conformational change when compared to Nsp15 without RNA present. As well the presence of RNA appeared to shift the electron density in Cryo-EM studies of Nsp15. This work advances the research in how Nsp15 may bind and cleave RNA and aid in the evasion of the host cell immune system.
ContributorsGoode, Matthew (Author) / Fromme, Petra (Thesis advisor) / Guo, Jia (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
Description
Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both a bacterial and a viral infectious disease in hopes of reducing infection, and consequently, fatality rates. The first protein of interest is OspA, a major outer surface protein in Borrelia burgdorferi – the causative bacterium of Lyme disease. Previous functional studies of OspA allude to both a role in colonization of B. burgdorferi in the tick vector and in evasion of the human immune system. This work describes the first ever structural studies of OspA as it is seen by the immune system: in the outer membrane. OspA was expressed in and purified from the outer membrane of Escherichia coli prior to characterization via circular dichroism (CD), native polyacrylamide gel electrophoresis, and electron microscopy. Characterization studies of OspA provide the first evidence of multimeric formation of OspA when translocated to the outer membrane, which presents a new perspective from which to build upon for the design of vaccinations against Lyme disease. The second protein of interest is nonstructural protein 15 (Nsp15), a protein responsible for facilitating immune system evasion of SARS-CoV-2 – the virus responsible for the COVID-19 pandemic. Nsp15 functions to enzymatically cleave negative sense viral RNA to avoid recognition by the human immune system. The work described in this dissertation is dedicated to the electron microscopy work utilized to reveal structural information on an inactive variant of Nsp15 bound to RNA sequences. Negative stain electron microscopy was used to verify Nsp15 structural integrity, as well as reveal a low-resolution image of structural deviation when RNA is bound to Nsp15. Cryo-electron microscopy was performed to solve structural density of Nsp15 without RNA to a resolution of 3.11 Å and Nsp15 bound to 5-nucleotides of RNA to a resolution of 3.99 Å. With further refinement, this structure will show the first structural data of Nsp15 bound to a visible RNA sequence, revealing information on the binding and enzymatic activity of Nsp15.
ContributorsKaschner, Emily (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra T (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
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
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
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually high number of predicted outer surface lipoproteins of unknown function but with multiple complex roles in pathogenesis, and an unusually low number of predicted outer membrane proteins, given the necessity of bringing in the required nutrients for pathogen survival. Cellular processing of bacterial membrane proteins is complex, and structures of proteins from Bb have all been solved without the N-terminal signal sequence that directs the protein to proper folding and placement in the membrane. This dissertation presents the first membrane-directed expression in E. coli of several Bb proteins involved in the pathogenesis of Lyme disease. For the first time, I present evidence that the predicted lipoprotein, BBA57, forms a large alpha-helical homo-multimeric complex in the OM, is soluble in several detergents, and purifiable. The purified BBA57 complex forms homogeneous, 10 nm-diameter particles, visible by negative stain electron microscopy. Two-dimensional class averages from negative stain images reveal the first low-resolution particle views, comprised of a ring of subunits with a plug on top, possibly forming a porin or channel. These results provide the first evidence to support our theories that some of the predicted lipoproteins in Bb form integral-complexes in the outer membrane, and require proper membrane integration to form functional proteins.
ContributorsRobertson, Karie (Author) / Hansen, Debra T. (Thesis advisor) / Fromme, Petra (Thesis advisor) / Van Horn, Wade (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020