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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Description
The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular biological factories with individualized subunits that function in concert to create novel peptides.One element at the heart of environmental health debates today is plastics. Biodegradable alternatives for petroleum-based plastics is a necessity. One NRPS, cyanophycin synthetase (CphA), can produce cyanophycin grana protein (CGP), a polymer composed of a poly-aspartic acid backbone with arginine side chains. The aspartic backbone has the potential to replace synthetic polyacrylate, although current production costs are prohibitive. In Chapter 2, a CphA variant from Tatumella morbirosei is characterized, that produces up to 3x more CGP than other known variants, and shows high iCGP specificity in both flask and bioreactor trials. Another CphA variant, this one from Acinetobacter baylyi, underwent rational protein design to create novel mutants. One, G217K, is 34% more productive than the wild type, while G163K produces a CGP with shorter chain lengths. The current structure refined from 4.4Å to 3.5Å.
Another exciting application of NRPSs is in healthcare. They can be used to generate novel peptides such as complex antibiotics. A recently discovered iterative polyketide synthase (IPTK), dubbed AlnB, produces an antibiotic called allenomycin. One of the modular subunits, a dehydratase named AlnB_DH, was crystallized to 2.45Å. Several mutations were created in multiple active site residues to help understand the functional mechanism of AlnB_DH. A preliminary holoenzyme AlnB structure at 3.8Å was generated although the large disorganized regions demonstrated an incomplete structure. It was found that chain length is the primary factor in driving dehydratase action within AlnB_DH, which helps lend understanding to this module.
ContributorsSwain, Kyle (Author) / Nannenga, Brent (Thesis advisor) / Nielsen, David (Committee member) / Mills, Jeremy (Committee member) / Seo, Eileen (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2022
Description
In recent years, researchers have employed DNA and protein nanotechnology to develop nanomaterials for applications in the fields of regenerative medicine, gene therapeutic, and materials science. In the current state of research, developing a biomimetic approach to fabricate an extracellular matrix (ECM)-like material has faced key challenges. The difficulty arises due to achieving spatiotemporal complexity that rivals the native ECM. Attempts to replicate the ECM using hydrogels have been limited in their ability to recapitulate its structural and functional properties. Moreover, the biological activities of the ECM, such as cell adhesion, proliferation, and differentiation, are mediated by ECM proteins and their interactions with cells, making it difficult to reproduce these activities in vitro.Thus, the work presented in my dissertation represents efforts to develop DNA and protein-based materials that mimic the biological properties of the ECM. The research involves the design, synthesis, and characterization of nanomaterials that exhibit unique physical, chemical, and mechanical properties. Two specific aspects of the biomimetic system have been to include (1) a modular protein building block to change the bioactivity of the system and (2) to temporally control the self-assembly of the protein nanofiber using different coiled coil mechanisms. The protein nanofibers were characterized using atomic force microscopy, transmission electron microscopy, and super-resolution DNA Point Accumulation for Imaging in Nanoscale Topology. The domains chosen are the fibronectin domains, Fn-III10, Fn-III9-10, and Fn-III12-14, with bioactivity such as cell adhesion and growth factor binding.
To extend this approach, these cys-nanofibers have been embedded in a hyaluronic acid scaffold to enable bioactivity and fibrous morphologies. Nanofiber integration within the HA gel has been shown to promote tunable mechanical properties and architectures, in addition to promoting a temporal display of the protein nanofibers. The hydrogels were characterized using scanning electron microscopy, mechanical compression testing, and fluorescence microscopy. The findings in this dissertation highlight the promise of biomimetic DNA and protein nanomaterials as a versatile approach for developing next-generation materials with unprecedented properties and functions. These findings continue to push the boundaries of what is possible in nanotechnology, leading to new discoveries that will have a significant impact on society.
ContributorsBernal-Chanchavac, Julio (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Jones, Anne (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
This thesis explores a diverse array of topics related to the role of dynamic allostery in regulating protein functions. Allostery is the phenomenon where a catalytic pocket responds to perturbations caused by binding at another distant site. This response often involves a conformational change resulting in a protein function alteration. However, it is essential to note the existence of dynamic allostery mechanisms that regulate protein function without relying on conformational changes but on dynamic motions. Within this thesis, position-specific equilibrium dynamics-based metrics like Dynamic Flexibility Index and Dynamic Coupling Index are employed to quantify the contributions of specific residues to protein dynamics. I investigated the role of dynamics in protein binding of the WW domain. In particular, I focused on how the mutations of distal positions modulate the binding site dynamics. By employing Dynamic Flexibility Index, I discovered that a residue, 10T, located distally from the binding pocket, plays a significant role in the observed dynamics difference between two variants: N21 (a native folded WW domain not binding Group I peptide) and CC16_N21 (an artificial WW domain binding Group I peptide). The T10H variant, created by exchanging the position 10 residue, enhances flexibility at positions 10 and 16. Consequently, this modification has led to an enhancement in the binding function of N21, enabling it to bind to Group I peptide effectively. Moreover, I investigated the influence of dynamic allostery on protein binding specificity, specifically in the PDZ domain PSD95. To gain insights into the binding process and accurately measure binding affinity, I employed two parallel computational approaches: Adaptive BP-docking and Steered Molecular Dynamics. These methods allowed me to model the binding interactions and quantify the binding strength robustly and comprehensively. The significance of allostery can serve as foundational knowledge in Deep Learning models, enabling the efficient mapping of protein sequences to their corresponding functionalities. One particular metric, Dynamic Coupling Index asymmetry, offers valuable insights into how the three-dimensional network of interactions facilitates communication within a protein structure. Leveraging these interactions, I developed a deep neural network architecture demonstrating enhanced capability in capturing epistatic interactions within Beta-lactamase and protein G function.
ContributorsLu, Jin (Author) / Ozkan, Banu (Thesis advisor) / Mills, Jeremy (Committee member) / Hariadi, Rizal (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2023
Description
Exoelectrogenic organisms transfer electrons from their quinone pool to extracellular acceptors over m-scale distances through appendages known as “biological nanowires”. These structures have been described as cytochrome-rich membrane extensions or pili. However, the components and mechanisms of this long-range electron transfer remain largely unknown. This dissertation describes supramolecular assembly of a tetraheme cytochrome into well-defined models of microbial nanowires and uses those structures to explore the mechanisms of ultra-long-range electron transfer. Chiral-induced-spin-selectivity through the cytochrome is also demonstrated. Nanowire extensions in Shewanella oneidensis have been hypothesized to transfer electrons via electron tunneling through proteinaceous structures that reinforce π-π stacking or through electron hopping via redox cofactors found along their lengths. To provide a model to evaluate the possibility of electron hopping along micron-scale distances, the first part of this dissertation describes the construction of a two-component, supramolecular nanostructure comprised of a small tetraheme cytochrome (STC) from Shewanella oneidensis fused to a peptide domain that self-assembles with a β-fibrillizing peptide. Structural and electrical characterization shows that the self-assembled protein fibers have dimensions relevant to understanding ultralong-range electron transfer and conduct electrons along their length via a cytochrome-mediated mechanism of electron transfer. The second part of this dissertations shows that a model three-component fiber construct based on charge complementary peptides and the redox protein can also be assembled. Structural and electrical characterization of the three-component structure also demonstrates desirable dimensions and electron conductivity along the length via a cytochrome-mediated mechanism.
In vivo, it has been hypothesized that cytochromes in the outer surface conduit are spin-selective. However, cytochromes in the periplasm of Shewanella oneidensis have not been shown to be spin selective, and the physiological impact of the chiral-induced-spin-selectivity (CISS) effect on microbial electron transport remains unclear. In the third part of this dissertation, investigations via spin polarization and a spin-dependent conduction study show that STC is spin selective, suggesting that spin selectivity may be an important factor in the electron transport efficiency of exoelectrogens.
In conclusion, this dissertation enables a better understanding of long-range electron transfer in bacterial nanowires and bioelectronic circuitry and offers suggestions for how to construct enhanced biosensors.
ContributorsNWACHUKWU, JUSTUS NMADUKA (Author) / Jones, Anne K. (Thesis advisor) / Mills, Jeremy (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2023
Description
Marine algae are a rich source of bioactive halogenated natural products. Thepresence of these marine natural products has largely been attributed to their biosynthesis
by organisms in these environments through a variety of different halogenation
mechanisms. One of the key contributors in these halogenation processes are from the
vanadium haloperoxidases (VHPOs) class of enzymes. VHPOs perform an electrophilic
halogenation through the oxidation of halide ions with hydrogen peroxide as the terminal
oxidant. This technique produces an electrophilic halide equivalent that can directly
halogenate organic substrates. Despite the numerous known reaction capabilities of this
enzyme class, their construction of intramolecular ring formation between a carbon and
nitrogen atom has remained unreported.
Herein, this study presents a development of a ‘new to nature’ chemical reaction for lactam synthesis. In pursuit of this type of reaction, it was discovered that wild type VHPOs (e.g., Curvularia inaequalis, Corallina officinalis, Corallina pilulifera, Acaryochloria marina) produce halogenated iminolactone compounds from acyclic amides in excellent yields and selectivity greater than 99 percent yield. The extension to chlorocyclizations will also be discussed.
ContributorsMerker, Kayla Rose (Author) / Biegasiewicz, Kyle (Thesis advisor) / Ackerman-Biegasiewicz, Laura (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2022
Description
G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level
structure of GPCRs facilitates the structure-based drug design. The most widely
used method currently for solving GPCR structure is still protein crystallography
especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native
environment of membrane protein which stable the membrane proteins. Traditional
synchrotron source requires large size large size protein crystals (>30 micron) due to
the radiation damage during data collection. However, acquiring large sized protein
crystals is challenging and not guaranteed practically. In this study, a novel method
was developed which combined LCP technology and micro-electron diffraction
(MicroED) technology. LCP-MicroED technology was able to collect complete
diffraction data sets from serval submicron protein crystals and deliver high
resolution protein structures. This technology was first confirmed with soluble
protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore,
this novel method was applied to a human GPCR target, Î22- adrenergic receptor
(Î22AR). The structure model was successfully built which proved the feasibility of
applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in
this research, a novel human GPCR target, human histamine 4 receptor(H4R) was
studied. Different constructs were expressed, purified, and characterized. Some key
residuals that affect ligand binding were confirmed.
ContributorsJing, Liang (Author) / Mazor, Yuval (Thesis advisor) / Mills, Jeremy (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2022
Description
Natures hardworking machines, proteins, are dynamic beings. Comprehending the role of dynamics in mediating allosteric effects is paramount to unraveling the intricate mechanisms underlying protein function and devising effective protein design strategies. Thus, the essential objective of this thesis is to elucidate ways to use protein dynamics based tools integrated with evolution and docking techniques to investigate the effect of distal allosteric mutations on protein function and further rationally design proteins. To this end, I first employed molecular dynamics (MD) simulations, Dynamic Flexibility Index (DFI) and Dynamic Coupling Index (DCI) on PICK1 PDZ, Butyrylcholinesterase (BChE), and Dihydrofolate reductase (DHFR) to uncover how these proteins utilize allostery to tune activity. Moreover, a new classification technique (“Controller”/“Controlled”) based on asymmetry in dynamic coupling is developed and applied to DHFR to elucidate the effect of allosteric mutations on enzyme activity. Subsequently, an MD driven dynamics design approach is applied on TEM-1 β-lactamase to tailor its activity against β-lactam antibiotics. New variants were created, and using a novel analytical approach called "dynamic distance analysis" (DDA) the degree of dynamic similarity between these variants were quantified. The experimentally confirmed results of these studies showed that the implementation of MD driven dynamics design holds significant potential for generating variants that can effectively modulate activity and stability. Finally, I introduced an evolutionary guided molecular dynamics driven protein design approach, integrated co-evolution and dynamic coupling (ICDC), to identify distal residues that modulate binding site dynamics through allosteric mechanisms. After validating the accuracy of ICDC with a complete mutational data set of β-lactamase, I applied it to Cyanovirin-N (CV-N) to identify allosteric positions and mutations that can modulate binding affinity. To further investigate the impact of mutations on the identified allosteric sites, I subjected putative mutants to binding analysis using Adaptive BP-Dock. Experimental validation of the computational predictions demonstrated the efficacy of integrating MD, DFI, DCI, and evolution to guide protein design. Ultimately, the research presented in this thesis demonstrates the effectiveness of using evolutionary guided molecular dynamics driven design alongside protein dynamics based tools to examine the significance of allosteric interactions and their influence on protein function.
ContributorsKazan, Ismail Can (Author) / Ozkan, Sefika Banu (Thesis advisor) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2023
Description
Since understanding the nature of proteins, it has been a long held belief that protein sequence dictated structure which then determined function. As such, all proteins contained structure and those that did not must not serve a purpose. For the last 25 years, scientists have begun to understand that disordered proteins, lacking structure, did not lack function. Their unique ability to undergo liquid-liquid phase separation served a cellular purpose, most involving nucleic acids. As more is uncovered, these unique proteins are being used to build new systems. Phase separated disordered proteins were used to design a functional organelle using the enzyme horseradish peroxidase and its chromatic substrate ABTS. Upon doing so, it was discovered that disordered proteins are highly susceptible to chemical modification through radical reactions with tyrosine. The increased frequency of tyrosine in disordered proteins provides multiple sites of conjugation by the ABTS radical and other substrates. These modifications then alter the physical properties of the proteins. The phase separated system was also incorporated with shell proteins from bacterial microcompartments in an attempt to limit access to the droplets. Through expression with truncations of the disordered sequence, shell proteins were able to interact with the droplets. Despite the appearance of complete coatings, they were found to be permeable to their surroundings, though much more stable than uncoated droplets. Just as disordered proteins are considered outside the traditional structures, so too are many students entering higher education. Non-traditional students are becoming more prevalent in the undergraduate population, though they are woefully underrepresented in the natural sciences. The benefits these students bring to their programs is highlighted and the circumstances that drive them away from STEM is explored. Non-traditional students contribute to the diversity of the scientific population, though many pursue education in non-STEM fields. To support these students, focus is put on andragogy (the teaching of adults), rather than pedagogy (the teaching of children). Non-traditional students face isolation and discrimination that is not being addressed by higher education institutions, hindering their ability to succeed. Through infrastructure designed for adult learners, STEM fields can be diversified in non-traditional ways.
ContributorsCostantino, Michele (Author) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy (Committee member) / Matyushov, Dmitry (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
Description
Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
ContributorsBarrett, Cassandra M (Author) / Haynes, Karmella A (Thesis advisor) / Rege, Kaushal (Committee member) / Mills, Jeremy (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2019