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- Genre: Academic theses
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- Status: Published
Description
In this study, the stability of two protein homo-oligomers, the β clamp (homodimer) from E. coli and the Proliferation Cell Nuclear Antigen (PCNA) from the yeast cell, were characterized. These clamps open through one interface by another protein called clamp loader, which helps it to encircle the DNA template strand. The β clamp protein binds with DNA polymerase and helps it to slide through the template strand and prevents its dissociation from the template strand. The questions need to be to answered in this research are, whether subunit stoichiometry contributes to the stability of the clamp proteins and how does the clamp loader open up the clamp, does it have to exert force on the clamp or does it take advantage of the dynamic behavior of the interface?
The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.
In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.
In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
ContributorsPUROHIT, ANIRBAN (Author) / Levitus, Marcia (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2020
Description
This thesis focuses on serial crystallography studies with X-ray free electron lasers
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
ContributorsKetawala, Gihan Kaushyal (Author) / Fromme, Petra (Thesis advisor) / Liu, Wei (Committee member) / Kirian, Richard (Committee member) / Arizona State University (Publisher)
Created2020
Description
Proteins are a large collection of biomolecules that orchestrate the vital
cellular processes of life. The last decade has witnessed dramatic advances in the
field of proteomics, which broadly include characterizing the composition, structure,
functions, interactions, and modifications of numerous proteins in biological systems,
and elucidating how the miscellaneous components collectively contribute to the
phenotypes associated with various disorders. Such large-scale proteomics studies
have steadily gained momentum with the evolution of diverse high-throughput
technologies. This work illustrates the development of novel high-throughput
proteomics platforms and their applications in translational and structural biology. In
Chapter 1, nucleic acid programmable protein arrays displaying the human
proteomes were applied to immunoprofiling of paired serum and cerebrospinal fluid
samples from patients with Alzheimer’s disease. This high-throughput
immunoproteomic approach allows us to investigate the global antibody responses
associated with Alzheimer’s disease and potentially identify the diagnostic
autoantibody biomarkers. In Chapter 2, a versatile proteomic pipeline based on the
baculovirus-insect cell expression system was established to enable high-throughput
gene cloning, protein production, in vivo crystallization and sample preparation for Xray diffraction. In conjunction with the advanced crystallography methods, this endto-end pipeline promises to substantially facilitate the protein structural
determination. In Chapter 3, modified nucleic acid programmable protein arrays
were developed and used for probing protein-protein interactions at the proteome
level. From the perspective of biomarker discovery, structural proteomics, and
protein interaction networks, this work demonstrated the power of high-throughput
proteomics technologies in myriad applications for proteome-scale structural,
functional, and biomedical research.
cellular processes of life. The last decade has witnessed dramatic advances in the
field of proteomics, which broadly include characterizing the composition, structure,
functions, interactions, and modifications of numerous proteins in biological systems,
and elucidating how the miscellaneous components collectively contribute to the
phenotypes associated with various disorders. Such large-scale proteomics studies
have steadily gained momentum with the evolution of diverse high-throughput
technologies. This work illustrates the development of novel high-throughput
proteomics platforms and their applications in translational and structural biology. In
Chapter 1, nucleic acid programmable protein arrays displaying the human
proteomes were applied to immunoprofiling of paired serum and cerebrospinal fluid
samples from patients with Alzheimer’s disease. This high-throughput
immunoproteomic approach allows us to investigate the global antibody responses
associated with Alzheimer’s disease and potentially identify the diagnostic
autoantibody biomarkers. In Chapter 2, a versatile proteomic pipeline based on the
baculovirus-insect cell expression system was established to enable high-throughput
gene cloning, protein production, in vivo crystallization and sample preparation for Xray diffraction. In conjunction with the advanced crystallography methods, this endto-end pipeline promises to substantially facilitate the protein structural
determination. In Chapter 3, modified nucleic acid programmable protein arrays
were developed and used for probing protein-protein interactions at the proteome
level. From the perspective of biomarker discovery, structural proteomics, and
protein interaction networks, this work demonstrated the power of high-throughput
proteomics technologies in myriad applications for proteome-scale structural,
functional, and biomedical research.
ContributorsTang, Yanyang (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen S (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2020
Description
Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ.
To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity.
To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies.
To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity.
To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies.
ContributorsNAZAROFF, CHRISTOPHER D. (Author) / Guo, Jia (Thesis advisor) / Rank, Matthew A (Thesis advisor) / LaBaer, Joshua (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2020
Description
Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future.
ContributorsShen, Di (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2020
Description
Glycans are complex biological sugar polymers that are commonly found covalently attached to proteins, lipids, and lipoproteins. About 50% of all mammalian proteins are glycosylated. Aberrant glycosylation is a hallmark of most types of cancer, and glycosylation changes that occur in this disease are known to facilitate tumor development. In this dissertation, a bottom-up approach to glycomics, “glycan node analysis”, which is a method based on glycan linkage analysis that quantifies unique glycan features, such as “core fucosylation”, “α2-6 sialylation”, “β1-6 branching”, and “bisecting GlcNAc”, as single analytical signals by gas chromatography-mass spectrometry (GC-MS), was applied to cancer cell lines, antibodies, extracellular vesicles, and low density lipoproteins to understand the mechanisms leading to aberrant glycosylation in cancer, and to understand the role of blood plasma glycan sialylation in cancer immunity. Specific tumor antigens such as β1-6-branching, β1-4-branching, bisecting GlcNAc, antennary fucosylation, and Tn antigen (GalNAc-Ser/Thr), were found to be regulated by IL-6 in HepG2 cells; fewer glycan features were regulated by IL-1β. Additionally, neuraminidase enzyme treatment of alpha-1 antitrypsin IgG demonstrates how glycan node analysis can be used to detect relative changes in “α2-6-sialylation” along with corresponding increases in terminal galactose.
Extracellular vesicles (EVs) derived from metastatic and non-metastatic cancer cell lines displayed upregulated or downregulated expression of several specific glycan nodes, particularly 3-GlcNAc, which represents hyaluronic acid. EVs displayed several glycan features that distinguished them from the whole blood plasma glycome. These results were promising for developing new diagnostic strategies in cancer.
A “liquid phase permethylation” procedure for glycan node analysis that does not require spin columns was applied for the first time to whole biological specimens, and it demonstrated potential clinical utility in detecting specific tumor antigens. Significantly different glycan node profiles were found among three cancer cell lines and in peripheral blood mononuclear cells from healthy donors.
Changes in glycosylation and mechanisms regulating glycan changes were studied extensively in cancer cells. Subsequently, it is reported how glycosylation changes can have an impact in cancer immunity. A novel role for oxidized-desialylated low density lipoprotein in cancer immunity is reported, and its implications in cancer and atherosclerosis are discussed.
ContributorsAguilar Diaz de leon, Jesús Salvador (Author) / Borges, Chad R (Thesis advisor) / Williams, Peter (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2021
Description
Staphylococcus aureus permanently asymptomatically colonizes one-third of humans, yet is an opportunistic pathogen causing life threatening diseases. Diagnosing S. aureus infections requires differentiating S. aureus from the human commensal Staphylococcus epidermidis, which beneficially colonizes the skin of all people. These studies aimed to characterize the volatile metabolites of S. aureus and S. epidermidis, and to measure the influence of growth medium on the discovery of volatile organic compounds that differentiate them. Headspace solid-phase microextraction and comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detected 337 S. aureus and S. epidermidis headspace volatiles produced during aerobic growth in four complex media. Analyses revealed that only 20 – 40% of staph volatiles are produced by both species in any one medium. Using principal components and hierarchical clustering analyses of the staphylococcal volatiles showed individual clustering of S. aureus and S. epidermidis independent of culturing media but clustering of replicate cultures by growth medium within species. Subsets of volatiles produced in common by both species, or in common across all four media, revealed volatilome differences between S. aureus and S. epidermidis based on the volatiles’ relative abundances. When analyzing volatiles by relative abundances, culturing staph in media containing free glucose (brain heart infusion and tryptic soy broth) revealed volatilomes dominated by acids and esters (67%). The low-glucose media (lysogeny broth and Mueller-Hinton broth) yielded ketones in greatest relative abundances, yet also produced highly dissimilar volatilome compositions. The staphylococcal volatilome is strongly influenced by the nutritional composition of growth medium, especially free glucose availability, which is robustly evident when analyzing the relative abundances of the volatiles, compared to their presence versus absence. Future work will evaluate more strains of each species, testing the universality of these results. Prospective analyses involve hypotheses testing on the role of catabolite repression control and glucose availability on the volatilome, with plans to model in vitro culture conditions that replicate in vivo volatilomes. Studies assessing correlations of virulence to species-specific volatilome responses to free glucose may identify pathogenic strains of S. epidermidis and other staphylococcal commensals.
ContributorsJenkins, Carrie L. (Author) / Bean, Heather D (Thesis advisor) / Buetow, Kenneth H (Committee member) / Lake, Douglas (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2021
Description
The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed and should ideally not use toxic reagents or create toxic by-products. The traditional methods of synthesizing nanoparticles generally are energy inefficient, requires stringent conditions such as high temperature, pressure or extreme pH and often produces toxic by-products. Although there exist a few solution-based methods to solve this problem, there is one avenue which has recently gained attention for nanoparticle synthesis: using biomolecules to facilitate nanomaterials synthesis. Using biomolecules for synthesis can provide a template to guide the nucleation process and helps to keep conditions biocompatible while also combining the step of functionalization of the nanoparticle with its synthesis through the biomolecule itself. The dissertation focuses on studying the bio-templated synthesis of two such noble metal nanoparticle which have biomedical applications: gold and platinum. In chapter 2, Gold Nanoparticles (GNP), with long-term stability, were synthesized using Maltose Binding Protein (MBP) as templating agent. The site of gold interaction on MBP was identified by X-ray crystallography. A novel gold binding peptide, AT1 (YPFGGSGGSGM), was designed based on the orientation of the residues in the gold binding site, identified through crystallography. This designed peptide was also shown to have stabilized and affected the growth rate of GNP formation, in similar manner to MBP. Further in chapter 3, a nanosensor was formulated using a variation of this GNP-MBP system, to detect and measure ionizing radiation dose for cancer radiation therapy. Upon exposure to therapeutic levels of ionizing radiation, the MBP‐based sensor system formed gold nanoparticles with a dose‐dependent color that could be used to predict the amount of delivered X‐ray dose. In chapter 4, a similar system of protein templated synthesis was introduced for platinum nanoparticle (PtNP). Here, GroEL, a large homo-tetradecamer chaperone from E.coli, was used as templating and stabilizing agent for reduction of K2PtCl4 ions to form PtNP. To understand how GroEL interacts with the PtNPs and thereby stabilizes them, single-particle cryo-electron microscopy technique was used to model the complex in solution. A 3.8-Å resolution 3D cryo-EM map of GroEL depicting the location of PtNP inside its central cylindrical cavity was obtained. Fitting a GroEL model to the map revealed Arginine-268 from two adjacent subunits of GroEL interacting with the PtNP surface. Finally in chapter 5, a solution to the potential issues of single particle data processing on protein nanoparticle complexes, specifically with 2D classification, was developed by creating masking algorithms.
ContributorsThaker, Amar Nilkamal (Author) / Nannenga, Brent L (Thesis advisor) / Acharya, Abhinav (Committee member) / Torres, Cesar (Committee member) / Mills, Jeremy (Committee member) / Rege, Kaushal (Committee member) / Arizona State University (Publisher)
Created2020
Description
Hydrogenase enzymes capable of catalyzing proton reduction to produce H2 have generated a considerable interest due to increasing motivation in finding sustainable carbon free energy sources. A considerable amount of research has been focused on producing synthetic structures mimicking the hydrogenase catalytic site, but the activity seen in hydrogenase enzymes in aqueous near neutral pH has yet to be replicated. It is now clear that the protein structure surrounding the H-cluster enables the high activity by fine tuning characteristics of the catalyst, but the structure and complexity of hydrogenase enzymes makes it difficult to predict exactly how the secondary coordination sphere affects catalysis. This work looks at incorporating both synthetic molecular catalysts and hydrogenase mimics into peptide scaffolds to improve the activity for photo-driven H2 production in aqueous solutions. The first chapter of this dissertation shows a de novo heme binding peptide improving the activity of cobalt protoporphyrin IX (CoPPIX) upon coordination inside a four-helix bundle. The peptide bound CoPPIX exhibited a 5.5-fold increase in anaerobic and an 8.3-fold increase in aerobic activity compared to free CoPPIX, while also showing dramatic increases to stability and solubility. In the second chapter, this work is expanded by using a randomly mutated cytochrome b562 library to identify beneficial attributes for downstream implementation of an ideal coordination site. A high-throughput assay was developed to measure H2 production using WO3/Pd deposited on a glass plate for a colorimetric first-pass screen. This assay successfully measured H2 production from CoPPIX bound cytochrome b562 in the periplasm of cells and identified a possible mutant showing 70% more H2 production compared to the wildtype. The third chapter incorporated a hydrogenase mimic into a four-helix bundle using a semi-synthetic strategy yielding a 3-fold increase in activity due to catalyst encapsulation. The method created will allow for easy modifications to the synthetic catalyst or peptide sequence in future work. The systems developed in this work were designed to facilitate the identification and implementation of beneficial characteristics for future development of an optimal secondary coordination sphere for a peptide bound molecular catalyst.
ContributorsHalloran, Nicholas Ryan (Author) / Ghirlanda, Giovanna (Thesis advisor) / Mills, Jeremy H (Committee member) / Moore, Gary F (Committee member) / Arizona State University (Publisher)
Created2021
Description
Since its conception over a century ago, X-ray crystallography (XRC) has become the most successful method used to elucidate the structures and functions of biological molecules at atomic resolution. The extensive use of XRC has led to meaningful discoveries across many scientific fields, notably its contributions to rational drug design. Traditional drug discovery relies on the use of trial-and-error based approaches in cellular and animal models of disease to identify chemical probes that elicit desirable therapeutic effects based off changes in phenotype. However, this approach lacks critical information in regards to the biological target in which the compound interacts with. In contrast, the use of rational drug design presents the opportunity to identify chemical probes that target specific protein targets of known medical importance and study their interactions using three dimensional structures that can be used to suggest new drug candidates. The main focus of my research presented in this dissertation aims to utilize XRC to discover novel therapeutics. In this work, I begin by describing the use of structure-based drug discovery for the rational design of hydrocarbon-stapled peptides that block Focal Adhesion Kinase (FAK) scaffolding in cancer (Chapter 2). FAK is an intracellular tyrosine kinase that has been linked to many cancers through its interaction with Paxillin LD motifs as it relates to tumor growth, invasion, metastasis, and suppression of apoptosis. The results of this study demonstrate the effectiveness hydrocarbon-stapling has on the native Paxillin LD2 sequence with ~50 fold greater binding affinity by surface plasmon resonance (SPR) that can be explained by the unique structural interactions observed by XRC. Next, I present a series of methods which lays the foundations for the discovery of novel anti-bacterial drugs that target 3-Deoxy-D-manno-octulosonate-8-phosphate (KDO8P) Synthase, a critical enzyme in the biosynthesis of gram-negative lipopolysaccharides (Chapter 3).
ContributorsThifault, Darren G. (Author) / Fromme, Petra (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021