Matching Items (40)
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Description
Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very important characteristic of Alzheimer’s is the accumulation of beta amyloid protein which often results in plaques. To understand these beta

Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very important characteristic of Alzheimer’s is the accumulation of beta amyloid protein which often results in plaques. To understand these beta amyloid proteins better, antibody fragments may be used to bind to these oligomers and potentially reduce the effects of Alzheimer’s disease.

This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
ContributorsColasurd, Paige (Author) / Nannenga, Brent (Thesis advisor) / Mills, Jeremy (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2018
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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

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.
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
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Description
Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer

Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.
ContributorsTekel, Stefan (Author) / Haynes, Karmella (Thesis advisor) / Mills, Jeremy (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2019
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Description
While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.
ContributorsAziz, Ann-Marie (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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Description
Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve

Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.
ContributorsDiep, Tristan H (Author) / Ghirlanda, Giovanna (Thesis director) / Redding, Kevin (Committee member) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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Description
G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to

G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.
ContributorsCosgrove, Steven Andrew (Author) / Liu, Wei (Thesis director) / Mills, Jeremy (Committee member) / Mazor, Yuval (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI complex, differ. In early evolving photoautotrophs, PSI<br/>exists in a trimeric organization, but in later evolving species this was lost and PSI exists solely<br/>as a monomer. While the reasons for a change in oligomerization are not fully understood, one<br/>of the 11 subunits within cyanobacterial PSI, PsaL, is thought to be involved in trimerization<br/>through the coordination of a calcium ion in an adjacent monomer. Recently published<br/>structures have demonstrated that PSI complexes are capable of trimerization without<br/>coordinating the calcium ion within PsaL.<br/>5 Here we explore the role the calcium ion plays in both<br/>the oligomeric and spectroscopic properties in PSI isolated from Synechocystis sp. PCC 6803.

ContributorsVanlandingham, Jackson R (Author) / Mazor, Yuval (Thesis director) / Mills, Jeremy (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description

Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields artificial enzymes that reduce protons to molecular hydrogen in the

Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields artificial enzymes that reduce protons to molecular hydrogen in the presence of photoinductive light and photosensitizers. Using random mutagenesis via error-prone PCR we have created a library of mutants to use in directed evolution to optimize hydrogen catalysis, though a challenge in this project is that testing individual variants by gas chromatography is not feasible on a large scale. For this reason, we are developing a gasochromic, hydrogen assay that is based on the interaction of molecular hydrogen with tungsten trioxide with a palladium catalyst. Initially, results show this assay to be qualitatively accurate between trials; however, its application in screening remains a challenge.

ContributorsGutierrez, Elijah (Author) / Ghirlanda, Giovana (Thesis director) / Mills, Jeremy (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05
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Description
Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has

Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.
ContributorsTruong, Chloe Du (Author) / Chiu, Po-Lin (Thesis advisor) / Liu, Wei (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
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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

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