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Browsing by Author "Fisk, Nick, committee member"

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    5-hydroxymethylcytosine and endonuclease G as regulators of homologous recombination
    (Colorado State University. Libraries, 2017) Vander Zanden, Crystal M., author; Ho, P. Shing, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Fisk, Nick, committee member
    Homologous recombination (HR) is a necessary biological process for all living organisms, and it is especially important for repairing damaged DNA. Improper HR results in DNA damage-related diseases, notably increased likelihood of cancer when HR regulators, such as the human BRCA1 gene, are impaired. HR is also a tool for biotechnology, giving scientists the power to easily delete or mutate genes and study the effects of those modifications. Recently, the epigenetically modified nucleotide 5-hydroxymethylcytosine (5hmC) was found to regulate vertebrate HR via interaction with the protein endonuclease G (EndoG). In this dissertation, I use biochemical/biophysical methods to elucidate the interaction between 5hmC and EndoG, thus working towards understanding their roles as regulators of recombination. I find that 5hmC forms a unique hydrogen bond to stabilize Holliday junctions, the four-stranded DNA intermediate in HR. 5hmC also induces a global structure change to the junction, increasing protein access to the junction crossover and providing potential for either direct or indirect readout of 5hmC. Further connecting EndoG with recombination, we present the first evidence that EndoG preferentially binds and cleaves Holliday junction DNA, implicating a role for EndoG as a resolvase. I demonstrate that EndoG recognizes 5hmC in the junction context and observe unique cleavage products from EndoG interaction with 5hmC-junctions. These results suggest that EndoG may have a previously unrecognized junction resolvase function and, in this way, play a more direct role in recombination than simply creating double-stranded breaks in duplex DNA to initiate the HR mechanism. Finally, I present a unique structural feature of vertebrate EndoG that we hypothesize is the basis for 5hmC recognition. I present the structure of mouse EndoG and propose that a two amino acid deletion, conserved in vertebrate EndoG sequences, is associated with unraveling of an α-helix. This structural perturbation positions amino acid side chains to confer 5hmC-sensing ability to all vertebrate EndoG. I expect that these deletion mutations and resulting structural effects co-evolved with the appearance of 5hmC in vertebrate genomes to give EndoG an additional function of recognizing 5hmC in the cell. Overall this work is building onto the understanding of 5hmC and EndoG as markers and regulators of recombination.
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    Catalytic biomass conversion and upgrading into platform chemicals and liquid fuels
    (Colorado State University. Libraries, 2014) Liu, Dajiang, author; James, Susan, advisor; Chen, Eugene, advisor; Williams, John, committee member; Marchese, Anthony, committee member; Fisk, Nick, committee member
    The development of novel, efficient catalytic processes for plant biomass conversion and upgrading into versatile platform chemicals as well as oxygenated biodiesel and premium hydrocarbon kerosene/jet fuels is described in this dissertation. The chief motivation of using annually renewable biomass as the source of chemical building blocks and transportation fuels is to reduce societal dependence on depleting fossil fuels. Towards this goal, 5-hydroxymethylfurfural (HMF), the dehydration product from C6 (poly)sugars, has been intensively investigated as it has been identified as a versatile C6 intermediate or platform for value-added chemicals and biofuels. This work has developed several highly efficient and cost-effective catalyst systems for C6 (poly)sugars conversion to HMF under mild conditions, including ubiquitous and inexpensive aluminum alkyl or alkoxy compounds, recyclable polymeric ionic liquid (PIL)-supported metal (Cr, Al) catalysts, and thiazolium chloride, a recyclable Vitamine B1 analog. An integrated, semi-continuous process for the HMF production from fructose has also been developed, affording the high-purity HMF as needle crystals. Towards HMF upgrading into higher-energy-density fuel intermediates, developing new strategies of C-C bond formation or chain extension is of particular interest. In this context, this study has discovered that N-heterocyclic carbenes (NHCs) are highly effective organic catalysts for HMF self-condensation to 5,5'-dihydroxymethylfuroin (DHMF), a new C12 biorefining building block. This new upgrading process has 100% atom economy, can be carried out under solvent-free conditions, and produces the C12 DHMF with quantitative selectivity and yield, the hallmarks of a "green" process. More significantly, the C12 DHMF has been transformed catalytically into oxygenated biodiesels, high-quality alkane jet fuels, and sustainable polymers, thereby establishing DHMF as a new C12 biomass platform chemical.
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    Generation of site-specific ubiquitinated histones through chemical ligation and characterization of histone deubiquitinases
    (Colorado State University. Libraries, 2016) Al-afaleq, Nouf Omar, author; Yao, Tingting, advisor; Cohen, Bob, committee member; Fisk, Nick, committee member; Peersen, Olve, committee member
    Nucleosome is the basic unit of chromatin and is composed of 147 base pairs of DNA wrapped 1.65 turns around a histone octamer of the four core histones (H2A, H2B, H3 and H4)(Luger et al., 1997). Histones are subject to numerous post-translational modifications. One such modification is the addition of a single ubiquitin (Ub) moiety to a specific lysine residue in the histones, such as H2AK119 or H2BK120 in humans. Depending on the site of Ub attachment, these modifications have distinct functional consequences. Whereas H2A ubiquitination is associated with transcriptional repression and silencing, H2B ubiquitination is associated with actively transcribed regions and has roles in initiation, elongation and mRNA processing. A more recently discovered ubiquitination site in H2A, H2AK13/15, is associated with DNA damage repair. In addition, a number of other ubiquitination sites on all types of histones have been discovered by high throughput mass spectrometry. The functions and regulations of those novel ubiquitinations are not known. Deubiquitinating enzymes (DUBs) reverse these ubiquitinations and therefore, are involved in a variety of regulatory processes. Mutations in several histone DUBs have been implicated in various diseases, thus they represent potential therapeutic targets. The specificity and regulation of histone DUBs are poorly understood in part because it has been difficult to obtain homogenous ubiquitinated histones and nucleosomes to use as substrates in vitro. Previously, several strategies have been developed to produce chemically defined ubiquitinated histones that use a combination of expressed protein ligation (EPL) and solid phase peptide synthesis (SPPS) techniques. These protocols are technically challenging for a biochemical lab. This dissertation describes our successful approach in obtaining homogenous site-specific ubiquitinated H2A and H2B that were then reconstituted into nucleosomes and used to qualitatively and quantitatively characterize a panel of known histone DUBs in vitro. We anticipate that our approach can be applied to generate all types of Ub-histone conjugates regardless of the particular ubiquitination site or histone types. They will significantly facilitate the study of all types of histone ubiquitination.
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    Isolation and characterization of a novel bacteriophage, ASC10, that lyses Francisella tularensis
    (Colorado State University. Libraries, 2014) Alharby, Abeer Mobsher, author; Gentry-Weeks, Claudia, advisor; Callahan, Gerald, committee member; Torsten, Eckstein, committee member; Fisk, Nick, committee member
    Francisella tularensis is an extremely infectious intracellular bacterium and the etiological agent of tularemia. Inhalation of Francisella tularensis can cause pulmonary tularemia, which has a mortality rate of 35% in the absence of treatment. Studies investigating the biology and molecular pathogenesis of Francisella tularensis have increased in the last few years especially after the U.S. Centers for Disease Control and Prevention (CDC) classified Francisella tularensis as a Category A Select Agent. In this dissertation, the identification and characterization of a novel temperate bacteriophage specific for Francisella species is described. Initial experiments focused on developing a media that would allow optimum growth of Francisella and recovery of bacteriophages. The preferred growth media for Francisella researchers is Mueller Hinton broth supplemented with IsoVitaleX enrichment mix. The research presented in this dissertation included development of a simple and low-cost brain heart infusion and Mueller Hinton base media, designated (BMFC), for enhancing the growth rate of all Francisella strains. BMFC media was compared with brain heart infusion media supplemented with cysteine (BHIc) for growth of Francisella tularensis subspecies novicida U112, Francisella tularensis subspecies holarctica LVS, and Francisella tularensis subspecies tularensis NR-50. Results from these experiments demonstrated that Francisella strains grow more rapidly when inoculated into BMFC media than when grown in BHIc media. A bacteriophage, designated ASC10, was discovered to be active against the majority of Francisella strains including F. tularensis Schu S4. This is the first bacteriophage reported to infect and lyse several Francisella species including F. tularensis subspecies holarctica strain LVS, F. tularensis subspecies tularensis strain Schu S4, F. philomiragia and F. t. novicida. Bacteriophage ASC10 was found to possess an icosahedral head of approximately 114 nm in diameter and a non-contractile tail of 92 nm in length. These measurements place ASC10 into the family of Siphoviridae. Bacteriophage ASC10 was isolated by mitomycin C induction of F. t. novicida NR-575 and plating of the phage lysate on F. t. novicida NR-584. Since these two strains are both mutants of F. t. novicida U112, it was hypothesized that they had genomic alterations that allowed F. t. novicida NR-584 to serve as a host for phage production. NextGen sequencing of the F. t. novicida NR-575 and NR-584 genomes and comparison by alignment revealed that F. t. novicida NR-584 had a 11.3 kb deletion encoding an abortive phage infection protein and a restriction-modification system. The location of the ASC10 prophage was identified within the Francisella novicida U112 genome by searching for homologous phage proteins using BLASTP. Based on significant similarity of clusters of phage-like proteins, it was determined that the ASC10 prophage is located between nucleotides 369,143 and 779,775. This location was chosen since it contained the integrase gene and other proteins associated with phage and was flanked by tRNA-serine loci. In summary, this dissertation describes the optimization of growth media for Francisella and the discovery of a unique phage that lyses Francisella species. The availability of a Francisella-specific bacteriophage is essential to develop rapid, field-deployable diagnostic assays, and would provide another tool in the arsenal for genetic manipulation of the Francisella genome.
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    Metabolic engineering and elucidation of the terpenoid indole alkaloid pathway in Catharanthus roseus hairy roots
    (Colorado State University. Libraries, 2016) Sun, Jiayi, author; Peebles, Christie A. M., advisor; Snow, Christopher D., committee member; Pilon-Smits, Elizabeth A. H., committee member; Fisk, Nick, committee member
    Catharanthus roseus (Madagascar periwinkle) produces many pharmaceutically important chemicals such as vinblastine, vincristine, serpentine, and ajmalicine. They are synthesized through the highly branched and complex terpenoid indole alkaloids (TIA) pathway in C. roseus. Among these TIAs, vinblastine and vinblastine, which are solely extracted from C. roseus, are the efficient anti-cancer drugs widely used in the clinic. However, due to the low accumulation of these TIAs within the plant and the industrial infeasibility of production using chemical synthesis, the market price of these drugs still remain high, and the production is inconsistent. With the advanced knowledge of molecular biology, metabolic engineering and bioinformatics, building a robust and efficient alternative production platform by manipulating the TIA pathway has become a major trend and promising strategy in recent research. However, many biosynthetic enzymes in TIA pathway and the regulation of the pathway are still poorly understood which impedes the rational engineering of this plant for enhanced TIA production. This thesis first uses advanced high-throughput sequencing technology to study the global transcriptional alterations after overexpressing a rate-limiting enzyme anthranilate synthase (AS) in the pathway. This study helps to increase understanding of TIA regulation in this transgenic hairy root line from a broader perspective. Furthermore, transcriptome sequencing of this unique transgenic line under three different conditions (uninduced control, induced AS overexpression, and methyl jasmonate elicitation) is analyzed using hierarchical clustering. A 200 candidate transcripts set was identified for the pathway genes located around the tabersonine branch point. Six cytochrome P450 monooxygenase candidates are selected for the unknown tabersonine 6,7 epoxidase that can convert tabersonine to lochnericine in C. roseus hairy roots. Meanwhile, effort on genetic modification of C. roseus hairy roots for TIA production using two different strategies are reported here. The first strategy helps establish a transgenic hairy root line with significantly increased TIA accumulation of all measure alkaloids by co-expressing the positive transcription factor ORCA3 (AP2-domain DNA-binding protein 3) and a pathway gene strictosidine glucosidase (SGD) that is not controlled by ORCA3. Since C. roseus hairy roots do not produce detectable vinblastine and vincristine due to the absence of the vindoline pathway, the second strategy initiated the effort to introduce the pathway by engineering the first two enzymes in C. roseus hairy root. Overexpression of these two genes, tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT), leads to the accumulation of the expected vindoline pathway intermediates 16-hydroxytabersonine and 16-methoxytabersonine but not vindoline. Interestingly, the overexpression of these two genes influences the root native metabolite levels, triggers the altered transcription of TIA genes, and leads to the production of two new unknown metabolites. Overall, studies in this thesis not only contribute new transcriptome information to current publicly available databases, but also facilitate elucidating the TIA pathway and its complex regulation. This thesis also provides a metabolic engineering approach to enhance alkaloid production in C. roseus hairy roots by simultaneously overexpressing ORCA3 and SGD. Genetic modification of T16H and 16OMT in C. roseus hairy roots promisingly leads to the production of vindoline pathway intermediates. It also emphasizes some potential complexities for the future attempts to express the full vindoline pathway in hairy roots.
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    Methods for detecting and developing protein-protein or protein-RNA interactions
    (Colorado State University. Libraries, 2014) Blakeley, Brett D., author; McNaughton, Brian, advisor; Kennan, Alan, committee member; Fisk, Nick, committee member; Reynolds, Melissa, committee member; Peersen, Olve, committee member
    Potent and selective recognition of disease-relevant macromolecules - such as proteins and RNA - is the molecular basis of most pharmaceuticals . Historically, small (< 500 Da) molecules have filled this role. However, the overwhelming majority (~85%) of the proteome - and emerging therapeutic targets such as RNA - present a serious challenge to small molecule-dependent recognition. An alternative approach to potent and selective recognition and regulation of disease-relevant proteins and RNA is to use synthetic proteins. In contrast to small molecules, the size, relatively high folding energies (>10 kcal/mol) and functional group diversity (by virtue of proteinaceous amino acids) allow proteins to recognize - and potentially control - macromolecular receptors that evade small molecules. Presented here are two approaches to advancing the discovery of new proteins that recognize either disease-relevant protein or RNA targets. The first part of this thesis describes split superpositive GFP reassembly as a method to identify novel protein-protein interacting pairs in living cells (E. coli). The second part of this thesis describes basic studies to evaluate the suitability of a naturally occurring RNA Recognition Motif (RRM) as a scaffold for targeting disease-relevant RNA hairpins, and the development of new RRMs that target TAR RNA, a hairpin critical to HIV proliferation.
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    Protein based technologies to identify, study, and control intracellular processes
    (Colorado State University. Libraries, 2017) Bruce, Virginia Jane, author; McNaughton, Brian, advisor; Ross, Eric, committee member; Di Pietro, Santiago, committee member; Fisk, Nick, committee member
    Proteins are increasingly used as basic research tools and therapeutics. Their large size, complex structure, and functional group diversity, by virtue of amino acids, often permit recognition of surfaces that challenge small-molecules. Fundamentally, this thesis describes protein based solutions to identifying macromolecules that function inside mammalian cells, enabling visualization and the study of complex biological processes in cellular environments. It also describes the development of engineered polycationic cell-penetrating nanobodies that access the cytosol and thus possibly represent a general solution to intracellularly targeted biologics drug discovery. Collectively, the work described in this thesis reports on: (1) a prostate cancer cell-selective cell penetrating peptide, and its optimization; (2) cell-penetrating nanobodies that access the cytosol of mammalian cells; (3) the use of engineered protein assemblies for bioanalytical reagents and to visualize transcription and translation in mammalian cells, and; (4) a new protein reassembly-based technology to measure cytosolic residence of intracellularly delivered proteins. Technologies and methods described in this work advance the use of proteins in basic science and therapeutically-relevant environments.
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    Protein resurfacing to identify macromolecular assemblies
    (Colorado State University. Libraries, 2016) Chapman, Alex Michael, author; McNaughton, Brian, advisor; Van Orden, Alan, committee member; Rovis, Tomislav, committee member; Fisk, Nick, committee member; Peersen, Olve, committee member
    Protein engineering is an emerging discipline that dovetails modern molecular biology techniques with high-throughput screening, laboratory evolution technologies, and computational approaches to modify sequence, structure, and in some cases, function and properties of proteins. The ultimate goal is to develop new proteins with improved or designer functions for use in biotechnology, medicine and basic research. One way to engineer proteins is to change their solvent exposed regions through focused or random 'protein resurfacing'. Here, I describe several approaches towards the development of synthetic proteins with new properties and function, including resistance to aggregation, increased solubility, and potent and selective macromolecule recognition. The first part of this thesis describes the use of protein supercharging to develop a split-superpositive GFP reassembly assay that is more efficient, faster, and more robust than previously described variants, largely due to increased resistance to aggregation. The second part of this thesis describes the use of shape complementarity, protein resurfacing, and high-throughput screening to evolve the first potent and selective protein-based inhibitor of the oncoprotein gankyrin. Concomitant with this work, I also describe a protein grafting strategy to identify a soluble mimic of S6 ATPase, which is subsequently used to characterize the S6 ATPase/gankyrin interaction by isothermal titration calorimetry.
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    Software for the use of protein fragment recombination and regression in protein structure determination and design
    (Colorado State University. Libraries, 2015) Lunt, Mark, author; Snow, Christopher, advisor; Fisk, Nick, committee member; Ben-Hur, Asa, committee member
    Recombination of protein structural fragments, in combination with regression-based scoring schemes, provides an alternative to existing iterative strategies for conducting a search over protein conformations. We developed software to define astronomically large combinatorial protein conformation search spaces, and to efficiently search those spaces. We demonstrate that such methods may be applicable to the structure prediction of cytochrome P450 chimeras. More generally, we demonstrate that such methods can be used to produce high-quality protein structural models given only low-resolution X-ray diffraction data.
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    Software to design crosslinks for protein crystal stabilization
    (Colorado State University. Libraries, 2015) Sebesta, Jacob Christopher, author; Snow, Christopher, advisor; Fisk, Nick, committee member; Rappe, Anthony, committee member
    Programmable materials allow properties as specific locations in the material to be modified through reliable encoding. One class of such materials are protein crystals that allow changes to be made through genetic manipulation. Protein crystals are well-ordered and highly porous materials, but they are also easily dissolved, limiting their utility. Crosslinking techniques previously developed often have a deleterious effects on the crystal order. In this work, we introduce software to design specific crosslinks across protein crystal interfaces using disulfide and dityrosine crosslinks as well as a variety of small molecule crosslinkers used in protein conjugation. The software is a general tool for specific crosslinking that introduces a number of improvements on previous disulfide design software. Several of the disulfide and dityrosine designs were assembled in the lab and one of the disulfide crosslink designs was confirmed using X-ray diffraction.
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    Towards macromolecular scaffold assisted crystallography
    (Colorado State University. Libraries, 2017) Huber, Thaddaus, author; Snow, Christopher, advisor; Ackerson, Christopher, committee member; Henry, Charles, committee member; Fisk, Nick, committee member
    The current, dominant method for structure determination in atomic detail is X-ray crystallography; but, this method requires a brute force search through non-physiological solution conditions looking for the "needle-in-a-haystack" condition in which the target protein crystallizes. Unfortunately, despite exhaustive screening, most proteins of interest do not form crystals. Other proteins are difficult to obtain in sufficient quantities to make the attempt. Finally, even successful crystals reveal a structure adopted under artificial conditions-a single snapshot that dramatically underrepresents the protein mobility. The motivational insight for this work is the recognition that materials diffract X-rays if they consist of a highly-ordered, repeating lattice, but that the lattice need not be composed only of target protein. Instead of growing conventional protein crystals, we will take the unprecedented step of attaching target proteins (guests) to specific sites within pre-existing, crystalline scaffolds for a new technique called scaffold assisted crystallography. This approach circumvents the haphazard nucleation and growth process that underlies conventional crystallography. Instead, we face novel challenges. We must engineer scaffolds that have very large pores (>10 nm), withstand significant solution condition changes, yet still diffract to high resolution. We must also ensure that guest proteins tightly tethered to the crystalline scaffold adopt a coherent structure visible via X-ray diffraction. Instead of taking on the challenge of de novo design of porous protein crystals, we decided to search the protein databank for a suitable scaffold. Algorithms for identifying highly porous protein crystals are covered (Chapter 1) and a select few representative examples are presented. Constructs for high priority candidates were obtained and crystallization of the targets were attempted. One of the candidates crystallized rapidly and presented a platform for developing methods and identifying roadblocks for second generation scaffolds. Working extensively with a single scaffold, a putative periplasmic protein from Campylobacter jejuni (CJ), allowed for robust method development that enabled highly optimized expression and extensive knowledge of its crystallization space. CJ requires high salt for crystallization. Crystals quickly degrade outside of the growth conditions. Most guest macromolecules will have low solubility in the high salt required to preserve the CJ crystalline lattice. Therefore, methods for chemical crosslinking of CJ crystals were developed to withstand significant solution condition changes, yet still diffract to high resolution. The most ubiquitous crosslinking agent glutaraldehyde effectively stabilized the crystal, but resulted in a dramatic loss of diffraction. Three alternative crosslinkers, formaldehyde, glyoxal, and EDC, were tested for their ability to stabilize CJ crystals. The three alternative crosslinkers all stabilized CJ crystals in challenging conditions (no salt) with little degradation in diffraction quality. The crosslinked crystals were subjected to x-ray diffraction; the resulting electron density demonstrates the first known atomic resolution modifications from formaldehyde, glyoxal, or EDC crosslinks in a protein crystal. In contrast to the weak, noncovalent interactions that hold together typical protein crystals, guest domains can be attached to the host scaffold using strong interactions. For maximum programmability, affinity tags for the desired assembly can be genetically encoded on the guest and scaffold monomers. We demonstrated that non-covalent, metal-mediated capture and genetically encoded histidine tags provide a significant level of control. Loading and release of guest molecules were fine-tuned to spatially segregate multiple guest proteins. Similarly, by controlling the diffusion of crosslinking agents we engineered a crystalline shell that still diffracts well. Scaffold assisted crystallography techniques were demonstrated with small molecule guests in CJ crystals. Guest molecules were installed via a single covalent bond to reduce the conformational freedom and achieve high occupancy structures. We used four different conjugation strategies to attach guest molecules to three different cysteine sites within pre-existing protein crystals. In all but one case, the presence of the adduct was obvious in the electron density. The above methods led to preliminary attempts of scaffold assisted crystallography with macromolecules. Guest mini-proteins variants were obtained with solvent exposed cysteines. These were covalently attached in vitro to CJ with an engineered surface thiol. We attempted to crystallize the resulting CJ-mini-protein conjugates. One of the CJ-mini-protein conjugates crystallized and the structure was determined. While the presence of the guest mini-protein was obvious, the electron density past the attachment point was ambiguous. Still, this result demonstrates feasibility of fusing target proteins to engineered CJ monomers for "chaperoned crystallization". For targets that fail to crystallize when pre-installed, we can perform asynchronous crystallization and by attaching the guest mini-protein to a preformed CJ crystal. Techniques for in crystallo conjugation and quantification are developed. Finally, present strategies for realizing macromolecular scaffold assisted crystallography are presented.