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.
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- All Subjects: Biochemistry
Description
The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.
ContributorsBowen, Sara (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2010
Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs.
Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
ContributorsNandi, Purbasha (Author) / Chiu, Po-Lin (Thesis advisor) / Mazor, Yuval (Committee member) / Hansen, Debra T (Committee member) / Arizona State University (Publisher)
Created2022
Description
The growing global energy demand coupled with the need for a low-carbon economy requires innovative solutions. Microalgal oxygenic photosynthesis provides a sustainable platform for efficient capture of sunlight and storage of some of the energy in the form of reduced carbon derivatives. Under certain conditions, the photosynthetic reductant can be shunted to molecular hydrogen production, yet the efficiency and longevity of such processes are insufficient. In this work, re-engineering of the heterodimeric type I reaction center, also known as photosystem I (PSI), in the green microalga Chlamydomonas reinhardtii was shown to dramatically change algal metabolism and improve photobiological hydrogen production in vivo. First, an internal fusion of the small PsaC subunit of PSI harboring the terminal photosynthetic electron transport chain cofactors with the endogenous algal hydrogenase 2 (HydA2) was demonstrated to assemble on the PSI core in vivo, albeit at ~15% the level of normal PSI accumulation, and make molecular hydrogen from water oxidation. Second, the more physiologically active algal endogenous hydrogenase 1 (HydA1) was fused to PsaC in a similar fashion, resulting in improved levels of accumulation (~75%). Both algal hydrogenases chimeras remained extremely oxygen sensitive and benefited from oxygen removal methods. On the example of PSI-HydA1 chimera, it was demonstrated that the active site of hydrogenase can be reactivated in vivo after complete inactivation by oxygen without the need for new polypeptide synthesis. Third, the hydrogenase domain of Megasphaera elsdenii bacterial hydrogenase (MeHydA) was also fused with psaC, resulting in expression of a PSI-hydrogenase chimera at ~25% the normal level. The heterologous hydrogenase chimera could be activated with the algal maturation system, despite only 32 % sequence identity (43 % similarity). All constructs demonstrated diminished ability to reduce PSI electron acceptors (ferredoxin and flavodoxin) in vitro and indirect evidence indicated that this was true in vivo as well. Finally, chimeric design considerations are discussed in light of the models generated by Alphafold2 and how could they be used to further optimize stability of the PSI-hydrogenase chimeric complexes.
ContributorsKanygin, Andrey (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Mazor, Yuval (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
To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given that menaquinone is active within the heliobacterial reaction center in some way. Purified heliobacterial reaction center was reconstituted into the liposomes and a recombinant cytochrome c553 was decorated onto the liposome surface. The native lipid-attachment sequence of cytochrome c553 was truncated and replaced with a hexahistidine tag. Thus, the membrane-anchoring observed in vivo was simulated through the histidine tag of the recombinant cytochrome binding to the Ni(II)-NTA lipid's head group. The kinetics of electron transfer in this system was measured and compared to native membranes using transient absorption spectroscopy. The preferential-orientation of reconstituted heliobacterial reaction center was also measured by monitoring the proteoliposome system's ability to reduce a soluble acceptor, flavodoxin, in both whole and detergent-solubilized proteoliposome conditions. These data demonstrate that this proteoliposome system is reliable, biomimetic, and efficient for selectively testing the function of the photosynthetic reaction center of Heliobacterium modesticaldum and its interactions with both donors and acceptors. The recombinant cytochrome c553 performs similarly to native cytochrome c553 in heliobacterial membranes. These data also support the hypothesis that the orientation of the reconstituted reaction center is inherently selective for its bacteriochlorophyll special pair directed to the outer-leaflet of the liposome.
ContributorsJohnson, William Alexander (Author) / Redding, Kevin E (Thesis advisor) / Van Horn, Wade D (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2018
Description
In my thesis, I characterize multi-nuclear manganese cofactors in modified reaction
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
ContributorsEspiritu, Eduardo (Author) / Allen, James P. (Thesis advisor) / Jones, Anne K (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2019
Description
The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
ContributorsGisriel, Christopher J (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2017
Description
The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that adds telomeric DNA repeats to the 3'-ends of chromosomes to offset the loss of terminal DNA repeats during DNA replication. It consists of two core components: a telomerase reverse transcriptase (TERT) and a telomerase RNA (TR). Telomerase uses a short sequence in its integral RNA component as template to add multiple DNA repeats in a processive manner. However, it remains unclear how the telomerase utilizes the short RNA template accurately and efficiently during DNA repeat synthesis. As previously reported human telomerase nucleotide synthesis arrests upon reaching the end of its RNA template by a unique template-embedded pause signal. In this study, I demonstrate pause signal remains active following template regeneration and inhibits the intrinsic processivity and rate of telomerase repeat addition. Furthermore, I have found that the human telomerase catalytic cycle comprises a crucial and slow incorporation of the first nucleotide after template translocation. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate, which is alleviated with elevated concentrations of dGTP. Additionally, molecular mechanism of the disease mutants on telomerase specific motif T, K570N, have been explored. Finally, I studied how telomerase selective inhibitor BIBR 1532 reduce telomerase repeat addition processivity by function assay. Together, these results shed new light on telomerase catalytic cycle and the importance of telomerase for biomedicine.
ContributorsChen, Yinnan (Author) / Chen, Julian J-L (Thesis advisor) / Jones, Anne K (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2018