Department of Biochemistry & Molecular Biology

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These digital collections include theses, dissertations, and datasets from the Department of Biochemistry & Molecular Biology.

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Open Access
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.
Open Access
A new dawn for Aurora B regulation: shining light on multiple discrete populations of Aurora B kinase at centromeres and kinetochores
(Colorado State University. Libraries, 2020) Broad, Amanda J., author; DeLuca, Jennifer G., advisor; Luger, Karolin, committee member; Markus, Steven, committee member; Yao, Tingting, committee member; Amberg, Greg, committee member
Cell division is a fundamental biological process that is essential for all eukaryotes to divide the replicated genome with high fidelity into individual daughter cells. Improper segregation of replicated DNA results in chromosome instability, a characteristic that is deleterious to most cells. Critical to the proper segregation of mitotic chromosomes is attachment to spindle microtubules, which are dynamic cytoskeleton filaments that drive the movement of chromosomes during mitosis. A complex network of proteins, collectively called the kinetochore, mediates microtubule attachments to chromosomes. Kinetochores are recruited to individual chromosomes through a specialized heterochromatin domain known as the centromere. Centromeric heterochromatin is comprised of both canonical, H3-containing nucleosomes as well as nucleosomes that contain the histone H3 variant CENP-A. Centromeres serve as a central point of organization in mitotic cells, recruiting both structural and regulatory kinetochore proteins to chromosomes. This extensive protein/DNA network ensures the accurate segregation of chromosomes by regulation of proper kinetochore-microtubule attachments in mitosis. Kinetochore-microtubule interactions are regulated by Aurora B kinase, which phosphorylates outer kinetochore substrates to promote release of erroneous attachments. Although Aurora B kinase substrates at the kinetochore are defined, little is known about how Aurora B is recruited to and evicted from kinetochores, in early and late mitosis, respectively, to regulate these essential interactions. We set out to determine how Aurora B kinase is regulated during mitosis. We found that, contrary to the current model, Aurora B kinase and the Chromosomal Passenger Complex are recruited to distinct regions within the centromere and kinetochore. Furthermore, we found that accumulation of Aurora B kinase at centromeres is independent from Aurora B localization and activity at outer kinetochores. These results lead us to hypothesize a new model for Aurora B kinase regulation. In the direct recruitment model, a population of the kinase is recruited directly to kinetochores in early mitosis, then as mitosis progresses and kinetochore-microtubule attachments are stabilized, architectural changes within the kinetochore result in the eviction of outer-kinetochore localized Aurora B kinase and the stabilization of kinetochore-microtubule attachments.
Open Access
A surface protease of Lyme disease bacteria degrades host extracellular matrix components and induces inflammatory cytokines in vitro
(Colorado State University. Libraries, 2012) Russell, Theresa Michelle Tidd, author; Bamburg, James R., advisor; Johnson, Barbara J. B., advisor; Luger, Karolin, committee member; Cohen, Robert E., committee member; Gentry-Weeks, Claudia, committee member
For nearly two decades, the paradigm in Lyme disease research has been that Borrelia burgdorferi does not produce proteases capable of damaging host molecules. Lyme disease has been considered, therefore, to be the consequence of an exuberant inflammatory response to infecting bacteria. This prevailing concept, however, has created a conundrum for the field. The bacterial burden in infected tissue is low, but the degree of inflammation is remarkable and seemingly out of proportion to this burden. The studies described in this dissertation provide evidence that, contrary to current thinking, B. burgdorferi does possess a protease that degrades numerous molecules of the host extracellular matrix (ECM). In addition to destabilization of the ECM which would be expected to benefit the organism, characterization of this proteolytic activity demonstrates that ECM fragments are produced that are known to be pro-inflammatory. These bioactive fragments may amplify the inflammatory processes triggered by the presence of the bacteria itself. When this hypothesis was tested directly by exposing chondrocytes to the borrelial protease in vitro, inflammatory cytokines and chemokines that are hallmarks of Lyme disease were induced. The studies herein suggest a new model for the pathogenesis of Lyme disease and offer an explanation for the paradox of debilitating inflammatory disease in the presence of few infecting organisms. Lastly, in contrast to current serology-based Lyme disease diagnostic tests, the activity of this protease in vitro may generate diagnostic biomarkers enabling detection of active B. burgdorferi infection.
Open Access
Activation of gene expression in yeast
(Colorado State University. Libraries, 2010) Lee, Sarah Kathleen, author; Stargell, Laurie Ann, 1963-, advisor; Luger, Karolin, committee member; Nerger, Janice Lee, 1960-, committee member; Nyborg, Jennifer Kay, committee member; Paule, Marvin R., 1943-, committee member; Ross, Eric D., committee member
Transcription is the generation of RNA from the DNA template, and is the fundamental aspect of gene expression. As such, the initiation of transcription at genes that are transcribed by RNA polymerase II (RNAPII) is a major control point in gene expression. Organisms across the evolutionary spectrum possess genes whose transcription is regulated after recruitment of RNAPII to the promoter, or postrecruitment. This regulatory strategy has been observed in bacteria, yeast, worms, flies, and humans. Therefore, postrecruitment regulation is a conserved strategy for controlling gene expression. Genome-wide studies in Drosophila and humans demonstrate that a significant portion of these genomes are postrecruitment regulated. Recent studies in humans indicate two biologically important activators (p53 and c-myc) are involved in releasing paused polymerases from promoter DNA1,2. These regulators of cell growth and differentiation are both implicated in carcinogenesis. Thus, further understanding how activators regulate the transition from an inactive to active polymerase will prove crucial in our understanding of transcriptional regulation and human diseases. Coactivators are conserved, multiprotein complexes involved in regulating the transcription process at most genes. Yet, virtually nothing is known about the role of coactivators at postrecruitment regulated genes in yeast. The work presented in this dissertation details the identification of postrecruitment functions of two coactivators, the Mediator and SAGA complexes. My studies reveal that coactivators act as intermediaries with activator proteins to stimulate transcription after the recruitment of RNAPII to the promoter. Further, this work demonstrates that this conserved class of factors plays a role in postrecruitment regulation, a previously unappreciated aspect of coactivator function. Analysis of Mediator function at the postrecruitment regulated CYC1 gene revealed a functional submodule of the Mediator complex that is required for triggering the preloaded polymerase at the CYC1 promoter into an active polymerase. This requirement exists even when two different activator proteins control CYC1 expression, Hap2/3/4/5 and Yap1. Strikingly, this submodule is not required for activation of a recruitment regulated Yap1-dependent gene, GTT2. The Yap1 activator controls the expression of a number of genes during oxidative stress in yeast. Oxidative stress is a damaging condition that haunts all aerobic organisms, and is linked to many human ailments. Yeast respond to this biological assault with a rapid activation of many genes. My investigation of Yap1-dependent transcription demonstrated that postrecruitment regulation is more prevalent in yeast than previously thought. Analysis of SAGA function at Yap1-dependent genes revealed that Yap1 utilizes SAGA during oxidative stress. Despite a common reliance on the SAGA coactivator for expression, each gene has different specific SAGA requirements. This demonstrates an important role for the SAGA coactivator during the important biological response to oxidative stress, and the complexity inherent in transcriptional regulation. In sum, my findings illustrate the mechanisms of activated transcription yeast utilize in response to important biological stimuli. This work significantly advances our understanding of the regulation of transcription after RNAPII arrives at the promoter. It also reveals the novel role that coactivators play in stimulating transcription at the group of genes that are regulated in this fashion.
Open Access
Adenosine triphosphate is an allosteric inhibitor of coxsackievirus B3 3Dpol
(Colorado State University. Libraries, 2016) Karr, Jonathan Paul, author; Peersen, Olve, advisor; Cohen, Robert, committee member; Perera, Rushika, committee member
Picornaviruses pose a significant threat to human and animal health, but at present there are no drugs to prevent or treat picornaviral infections. However, intensive study of the picornaviral lifecycle has revealed several promising pharmacological targets, including the RNA-dependent RNA polymerase, 3Dpol, that is responsible for replicating the viral genome. 3Dpol is central in the virus lifecycle, determines the distribution of mutants in viral progeny, and is a very highly conserved protein across picornavirus species. As such, it is an attractive target for antiviral research. The only 3Dpol inhibitors that have been found to date are nucleoside analogues that act directly on the active site, even though global dynamics of the protein that are sensitive to allosteric effects of various mutations have been shown to be important determinants of fidelity. The research presented in this thesis provides the first direct evidence of allosteric regulation of 3Dpol by a small molecule. Inhibition assays investigating the relative affinities of a stalled coxsackievirus elongation complex for non-cognate nucleotides uncovered a mixed inhibition profile of ATP. Among the six picornavirus species tested, this mode of inhibition seems specific to coxsackievirus B3 (CVB3). Engineered mutations in CVB3 3Dpol, including two that were previously found to lower polymerase fidelity, diminish the uncompetitive component of ATP inhibition. ATP inhibition was found to be dependent on the β- and γ-phosphates. The potential role of ATP’s allosteric effect in the virus lifecycle as well as the importance of a biochemically confirmed allosteric site on the polymerase are discussed.
Open Access
Archaeal isoprenoid biosynthesis
(Colorado State University. Libraries, 2018) Liman, Lie Stefanus Geraldy, author; Santangelo, Thomas, advisor; Laybourn, Paul, committee member; Peebles, Christie, committee member
Many high value natural products - including artemisinin, squalene, and farnesene – are isoprenoids. Efforts to commercially produce isoprenoids are often complicated by low concentrations of isoprenoid precursors and the toxicity of isoprenoids in common production platforms (i.e. bacteria and yeasts). Archaeal-based production platforms provide a potential solution to the precursor toxicity problems as archaea produce isoprenoids in large quantities to generate their unique membrane hydrocarbon chains. One roadblock to commercial archaeal isoprenoid production platforms is the uncharacterized pathway leading to isoprenoid precursor synthesis. This project details, genetically and biochemical, the first three steps in the proposed pathway of archaeal isoprenoid biosynthesis - from acetyl-CoA to mevalonate - in Thermococcus kodakarensis.
Open Access
Archaeal transcription and replication: new insights into transcription-coupled DNA repair and origin-independent DNA replication
(Colorado State University. Libraries, 2017) Gehring, Alexandra Marie, author; Santangelo, Thomas, advisor; Argueso, J. Lucas, committee member; Nyborg, Jennifer K., committee member; Peersen, Olve B., committee member
The three Domains of extant life use similar mechanisms for information processing systems. Although many aspects of replication, transcription and translation are universally conserved, the evolutionary history of the enzymes involved is not always clear and domain-specific differences are known. The transcription apparatus, especially the multi-subunit RNA polymerase (RNAP), has a clear evolutionary conservation across all Domains. Elucidating the mechanisms of the transcription apparatus in Archaea will help further understanding of underlying transcription mechanisms and regulation of those mechanisms, not only in Archaea but also in Bacteria and Eukarya. Conversely, the DNA replication machinery, most notably the replicative DNA polymerases, are distinct for each Domain. Any demonstration of the activities of the replication proteins, and especially discovery of unique pathways and mechanisms underlying replication helps to improve the understanding of the larger evolutionary questions surrounding DNA replication. The compact nature of archaeal genomes necessitates timely termination of transcription to prevent continued transcription of neighboring genes while ensuring complete transcription of the gene of interest. Transcription elongation is processive, and the transcription elongation complex is exceptionally stable. The disruption of this transcription elongation process, transcription termination, is an essential step in the transcription cycle. The presence of DNA lesions causes early termination of transcription in Bacteria and Eukarya. The results of this dissertation demonstrate this is also true in Archaea. Archaeal RNAP arrests transcription at DNA lesions and likely initiates transcription-coupled DNA repair (TCR) as will be soon demonstrated using in vivo techniques developed during this dissertation work. DNA replication is a highly regulated cellular process, particularly initiation of DNA replication. The long-standing replicon hypothesis states a trans-acting replication initiation protein must recognize a cis-acting DNA element, the origin of replication. For the 50 years after the replicon hypothesis was first posited, the replication hypothesis was supported in phages, Bacteria, Archaea, and Eukarya. The work presented in this dissertation describes the non-essentiality of Cdc6 and the origin of replication, and further demonstrates that origin-independent DNA replication is the mechanism by which Thermococcus kodakarensis replicates its genome. The results of this study and others in the field brings forward questions about the evolutionary history of DNA replication in all three Domains of extant life.
Open Access
Aromatic circular dichroism in globular proteins: applications to protein structure and folding
(Colorado State University. Libraries, 1994) Grishina, Irina Borisovna, author; Woody, Robert W., advisor
The exciton couplet approach was applied to estimate the circular dichroism (CD) of Trp side-chains in proteins. Calculations were performed by the origin-independent version of the matrix method, either for the indole Bb transition only or for the six lowest energy indole transitions. The dependence of the CD of a Trp pair upon its distance and geometry has been analyzed. It was predicted that mixing with far-uv transitions are as important in determining the CD intensity of the near-uv transitions as the coupling among near-uv transition. The effects of varying exposure of Trp chromophores and nearby charges on Trp CD have been examined. A survey of a large number of proteins from the Protein Data Bank reveals a number of cases where readily detectable exciton couplets are predicted to result from the exciton coupling of Trp Bb bands. The predicted CD spectra are generally couplets, often dominated by the contributions of the closest pair, but sometimes exhibit three distinct maxima. This CD depends on the distance and relative orientation of Trp pairs and thus reflects the spatial arrangement of Trp residues in the protein. It was shown that Trp side chains can make significant contributions to the CD of proteins in the far ultraviolet. The distance dependence of exciton splitting, rotational and couplet strengths of Trp pairs show general agreement with theoretical predictions. In several cases, changes in protein Trp CD can be attributed to a specific Trp pair and explained as a definite change in its conformation. Applications of the exciton couplet approach are discussed for various crystal forms of hen lysozyme, turkey and human lysozyme. Trp62 in hen and turkey lysozymes was found to be sensitive to the perturbations of the protein surface due to binding of substrate, antibodies and intermolecular contacts in the crystal. Conformational changes of Trp62 are predicted to have a strong effect on the overall Trp CD of lysozyme. Predicted Trp CD is compared with experimental results for various lysozymes, a-chymotrypsin and chymotrypsinogen A, concanavalin, dihydrofolate reductase and ribonuclease from Bacillus intermedius 7P (binase). The calculated near-uv CD for hen lysozyme matches the experimental amplitude. Correlation of conformational changes in proteins with Trp CD is shown for a-chymotrypsin and chymotrypsinogen A. We found that the exciton couplet approach might be useful in relating Trp CD and changes in protein structure upon local mutations and conformational changes involved in enzyme activation. Small globular proteins are usually composed of a single structural domain and undergo cooperative denaturation. We have demonstrated that a protein with a single structural domain, binase, and a protein with multiple structural domains, porcine pepsin, contain fewer cooperative regions (energetic domains) under the conditions optimal for their functional activity. The study was performed by combining a CD analysis of the structural changes in the proteins during thermal denaturation and under various solvent conditions with thermodynamic properties observed by scanning microcalorimetry. Estimates of secondary structure were obtained from CD spectra, taking side-chain CD into account. It was found that neither of the proteins show any changes in secondary structure or local environment of aromatic amino acids upon separation of the energetic domains. The structural regions in binase corresponding to energetic domains were identified. It was shown that binase is converted from a single cooperative system into two separate energetic domains when ion pairs are disrupted, whereas the size of cooperative units in pepsin decrease as the electrostatic repulsion between regions in the molecule increases.
Open Access
Assessing histone H2A.Z and the H2A tails in chromatin structure
(Colorado State University. Libraries, 2018) Seidel, Erik, author; Hansen, Jeffrey, advisor; Stargell, Laurie, committee member; Bailey, Susan, committee member
Deoxyribose nucleic acid (DNA) is a negatively charged macromolecule that encodes life's genetic material. In organisms, it is bound to net positively charged histone proteins in specific fashions and then compacts with magnesium and calcium to form domains and then chromosomes, which occupy territories in the nucleus during interphase. The mechanism of this compaction has been debated and studied for decades, and the employment of specific protein structures in molding chromatin morphology is still under review. This thesis adds to this story by testing how higher order chromatin structure is influenced by a histone H2A variant, H2A.Z, and the combined effect that the so-called histone H2A N and C terminal tails, when contrasted to arrays involving wildtype canonical H2A. An in vitro model system of nucleosomal arrays consisting of sea urchin derived 5S ribosomal DNA and recombinant mammalian histone proteins was used. Both the H2A.Z and H2A tailless arrays required increased magnesium to oligomerize into possibly domain-like structures. The H2A.Z protein produced similarly accessible structures as the fully accessible wildtype control as learned through a micrococcal nuclease digestion method designed for these chromatin structures. The deletion of the H2A N and C terminal tails produced oligomers with slightly less accessible linker DNA than its wildtype control according to the micrococcal nuclease digestion. Furthermore, the H2A.Z, H2A double tailless, and H2A wildtype oligomers were globular in shape. When subjected to fluorescence recovery after photobleaching (FRAP), the oligomers involving H2A.Z agreed with the current literature describing its presence in euchromatin and heterochromatin, and its mobility correlated with that of a more mobile and possibly more open structural agent. Taken together, the H2A and H2A.Z proteins are influential in determining and providing variability to the overall chromatin structure that is vital to DNA's role in biology.
Open Access
Aurora A kinase phosphorylates serine 62 on Hec1 to affect mitotic kinetochore microtubule interactions
(Colorado State University. Libraries, 2024) Sparrow, Sarah, author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Bailey, Susan, committee member
The Hec1 protein plays an important role in ensuring successful chromosome segregation during cell division. Its 80 amino acid, unstructured, "tail" region is critical for kinetochore-microtubule attachment regulation, which is mediated through Aurora kinase phosphorylation. At least nine phosphorylation target sites within this domain have been identified, including the recently confirmed target site, serine 62 (S62). However, the functional significance of phosphorylation of this residue remains elusive. Here, we selectively target Aurora A and Aurora B kinase protein activities using the inhibitors MLN8054 and ZM447439, respectively, and study their effects on the dynamics of serine 62 phosphorylation in the Hec1 tail. Utilizing immunofluorescence, we demonstrated that inhibition of Aurora A kinase activity leads to a significant reduction in phosphorylation levels at serine 62. Additionally, using phospho-null mutants, we studied the effect of serine 62 phosphorylation on the creation of stable, tension-generating kinetochore-microtubule attachments by measuring the distance between sister kinetochores. Our findings reveal that alterations in serine 62 phosphorylation status result in subtle changes in interkinetochore distances showcasing the functional relevance of this phosphorylation event in regulating kinetochore-microtubule attachments. Furthermore, under conditions of nocodazole-induced mitotic arrest, we observe a marked decrease in phosphorylation at serine 62 suggesting a microtubule dependent regulation of this phosphorylation. These findings provide evidence supporting the role of Aurora A kinase in phosphorylating serine 62 of the Hec1 tail and shed light on the regulation of this critical post-translational modification during mitosis.
Open Access
Biochemical, biophysical and functional characterization of histone chaperones
(Colorado State University. Libraries, 2014) Zhang, Ling, author; Luger, Karolin, advisor; Krapf, Diego, committee member; Nyborg, Jennifer, committee member; van Orden, Alan, committee member; Stargell, Laurie, committee member
Nucleosomes, the basic repeating unit of chromatin, are highly dynamic. Nucleosome dynamics allow for various cellular activities such as replication, recombination, transcription and DNA repair, while maintaining a high degree of DNA compaction. Each nucleosome is composed of 147 bp DNA wrapping around a histone octamer. Histone chaperones interact with histones and regulate nucleosome assembly and disassembly in the absence of ATP. To understand how nucleosome dynamics are regulated, it is essential to characterize the functions of histone chaperones. The first project of my doctoral research focused on the comparison of different nucleosome assembly proteins employing various biochemical and molecular approaches. Nucleosome assembly proteins (Nap) are a large family of histone chaperones, including Nap1 and Vps75 in Saccharomyces cerevisiae, and Nap1 (also Nap1L1), Nap1L2-6 (Nap1-like 2-6, with Nap1L4 being Nap2) and Set in metazoans. The functional differences of nucleosome assembly proteins are thus interesting to explore. We show that Nap1, Nap2 and Set bind to histones with similar and high affinities, but Nap2 and Set do not disassemble non-nucleosomal DNA-histone complexes as efficiently as Nap1. Also, nucleosome assembly proteins do not display discrepancies for histone variants or different DNA sequences. In the second project, we identified Spn1 as a novel histone chaperone and look into new functions of Spn1 on the regulation of chromatin structural states. Spn1 was identified as a transcription regulator that regulates post-recruitment of RNA polymerase II in yeast. We demonstrated that Spn1 is a H3/H4 histone chaperone, a novel finding that was not observed previously. Spn1 also interacts with Nap1, and forms ternary complexes with Nap1 and histones. We also show that Spn1 has chromatin assembly activity and N- and C- terminal domains of Spn1 are required for its histone chaperone properties. At the same time, we had an interesting observation that Spn1 potentially has topoisomerase/nuclease activity, which is dependent on magnesium ions. This activity of Spn1 can also help answer questions raised by in vivo assays related to Spn1, including its correlation with telomere length, the heat sensitivity in the reduction of function yeast strains, and the elongated lifespan in the Spn1ΔNΔC strain. Our studies on the functional comparison of nucleosome assembly proteins revealed their distinct roles in the regulation of nucleosome dynamics. Our findings on the histone chaperone functions and nuclease/topoisomerase activities disclosed new roles of Spn1 in chromatin regulation, by regulating histone-DNA interaction and also maintenance of DNA integrity.
Open Access
Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex
(Colorado State University. Libraries, 2009) Yang, Chenghua, author; Luger, Karolin, advisor
Methyl-CpG Binding Protein (MeCP2) is an abundant chromatin associated protein that is important in maintaining human health; mutations in this protein cause Rett Syndrome, a neurodevelopmental disease that is a common cause of mental retardation and autism in females. MeCP2 was initially identified as a protein that recognizes the genetic DNA methyl-CpG mark and it was thought to repress gene transcription by recruiting histone deacetylases. Recent studies show that MeCP2 can both repress and activate gene transcription. It also binds chromatin in the absence of the methylation mark, suggesting that its mode of action is more complex than previously assumed. The observation that MeCP2 compacts nucleosomal arrays in vitro and mediates silent chromatin loop formation in vivo suggests a novel mechanism by which MeCP2 regulates gene expression. To further characterize the interplay between MeCP2 and chromatin, it is important to understand the interactions between MeCP2 and nucleosomes, the fundamental component of chromatin. We used biochemical and biophysical approaches to study the interplay between MeCP2 and nucleosomes. Gel mobility assays showed that although MeCP2 can interact with a nucleosome with or without extra nucleosomal DNA, it has a higher affinity for nucleosomes with extra nucleosomal DNA. The N-terminal portion of human MeCP2 (amino acids 78-305) is sufficient to establish this interaction. Size-exclusion chromatography combined with multi-angle light scattering and fluoresecence resonance energy transfer (FRET) assays demonstrated that this interaction occurs at a 1:1 molar ratio and that MeCP2 brings the extra nucleosomal DNA ends in a closer proximity. Small angle X-ray scattering (SAXS) revealed the formation of a more compact complex when MeCP2 interacts with nucleosome with (versus without) extra nucleosomal DNA, indicating that the extra nucleosomal DNA is important in organizing the MeCP2-nucleosome complex. Our data suggest a model in which MeCP2 compacts chromatin by changing the extra nucleosomal DNA path. X-ray crystallography is also used to characterize the nucleosome-MeCP2 complex. Crystals of the nucleosomes with extra nucleosomal DNA in complex with MeCP2 were obtained and diffracted to 5.2 Å. Although MeCP2 dissociated from the crystals after soaking in cryo-protectant, the electron density map reveals the path of extra nucleosomal DNA which may be organized by MeCP2.
Open Access
Biophysical, structural, and functional studies of histone binding proteins
(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
Open Access
Cargo induced recruitment of the endocytic adaptor Sla1 and the role of Sla1-clathrin binding in endocytosis
(Colorado State University. Libraries, 2018) Tolsma, Thomas O., author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Reist, Noreen, committee member
Clathrin-mediated endocytosis is a highly dynamic process that is essential in all eukaryotes. This process is utilized for a number of functions including the uptake of extracellular nutrients, manipulation of the plasma membrane content, downregulation of cell signaling pathways, and viral entry. While differences in protein composition, sequence, and structure do exist between species for this process, many core protein functions and the mechanistic steps involved in endocytic vesicle formation and internalization are highly conserved. This has allowed findings from one species to be applicable to another. For this reason Saccharomyces cerevisiae has been characterized as a highly useful model organism for studying and identifying key proteins and conserved mechanisms in clathrin-mediated endocytosis that are found in all eukaryotes. In yeast, roughly 60 proteins have been identified as being part of the endocytic machinery. Clathrin-mediated endocytosis begins with the recruitment of early endocytic proteins that establish the site of endocytosis. This includes scaffolding and coat proteins, such as clathrin, that aggregate at the plasma membrane through interactions with lipids, protein cargo, and other components of the endocytic machinery. This is followed by recruitment of other late coat proteins that further prepare the site for internalization. Following coat formation the mobile phase of membrane invagination is initiated by the recruitment of the actin polymerization machinery. Actin polymerization then generates an inward force at the site of endocytosis that causes invagination of the plasma membrane. The invagination is then separated from the plasma membrane through the recruitment of scission proteins that pinch off the endocytic vesicle. Lastly the internalized vesicle undergoes a process of coat protein disassembly before being targeted to its proper destination in the cell. While much of this process has been well characterized, significant gaps in our understanding of how different steps in endocytic progression are coordinated and how endocytic proteins function still exist. Using a combination of yeast genetics, fluorescent microscopy, electron microscopy, and biochemistry we have furthered our understanding of clathrin-mediated endocytosis, focusing on the role adaptor-clathrin and adaptor-cargo binding plays in formation and progression of the endocytic process. Our work began by focusing on the role of the adaptor protein Sla1, a clathrin and cargo binding protein that serves essential roles in endocytosis. It was previously established that Sla1 binds clathrin through a variable clathrin box of sequence LLDLQ. Loss of clathrin binding by mutation of this clathrin box has a dramatic effect on endocytosis such as an increased patch lifetime of Sla1 at endocytic sites, and dramatic defects in internalization of endocytic protein cargo. While these experiments demonstrated the importance of Sla1-clathrin binding in endocytosis, they did not explain why Sla1-clathrin binding was important and how this interaction contributes mechanistically to endocytic progression. By imaging Sla1 and clathrin, our work demonstrates that Sla1 contributes to proper clathrin recruitment to endocytic sites. A loss of proper recruitment of clathrin to endocytic sites by mutation of the Sla1 variable clathrin box also resulted in significant accumulation of other endocytic proteins, including those involved in actin polymerization. The lifetime of these additional endocytic components lasted for significantly longer at endocytic sites, some having a disruption in normal recruitment dynamics. Despite this accumulation of the actin polymerization machinery, there is a significant delay in actin polymerization and an increase in actin polymerization time and levels at endocytic sites. Our results also demonstrate defects in the formation of the endocytic invagination and delays in scission. Thus, the Sla1-clathrin interaction is needed for normal progression through different stages of the endocytic process. A second question in the endocytic field that has received little attention is the role cargo plays in the recruitment of the endocytic machinery. The conventional view is that first the endocytic machinery forms an endocytic site and then cargo is concentrated by binding adaptor proteins. Sla1 has previously been shown to bind to endocytic protein cargo that contains the amino acid sequence NPFxD through its SHD1 domain. It has also been shown through biochemical experiments that Sla1 binds Ubiquitin via its third SH3 domain. Both NPFxD and Ubiquitin have been shown to be important signals of cargo for entry into the endocytic pathway. The question, however, remained as to whether cargo binding via these signals contributes to recruitment of the adaptor Sla1 to endocytic sites. The work described in this dissertation will present evidence that this is indeed the case.
Open Access
Characterization of HIV-1 protease autoprocessing trans-cleavage mechanism
(Colorado State University. Libraries, 2014) Machihara, Satoshi, author; Chen, Chaoping, advisor; Ross, Eric, committee member; Rovnak, Joel, committee member
HIV protease is an aspartic acid enzyme responsible for the cleavage reactions essential in the maturation (infectivity) of the viral particle. Protease inhibitors (non-cleavable substrate analogs) have been potent tools in combating HIV infection as well as its result - AIDS. However, the emergence of drug-resistant viruses in patients treated with these inhibitors is an ongoing concern. Thus there is a growing need to find additional therapeutic targets and treatments to supplement the currently available protease inhibitors. A promising new target for drug development is protease autoprocessing which is a virus-specific process responsible for the release of the mature protease from its precursor (Gag-Pol). Unfortunately, structural and mechanistic information pertaining to autoprocessing are yet insufficient. According to the mature protease structure, it is speculated that precursor dimerization is essential for autoprocessing to occur. We have developed a model system to specifically examine the trans-cleavage mechanism mediated by engineered fusion precursors (differentially labeled substrate and enzyme, respectively). Using this system, we demonstrate that trans-cleavage happens between fusion precursors both in the presence and absence of a dimer inducing fusion tag (DIFT). Trans-cleavage was also observed when monomeric fusion tags were attached to the fusion precursor. These results hint that autoprocessing mediated by the fusion precursor is independent of dimer-inducing tag in our model system.
Open Access
Characterization of interactions of lipoquinone derivatives within model membrane systems
(Colorado State University. Libraries, 2021) Bublitz, Gaia Rachel, author; Crans, Debbie, advisor; Cohen, Robert, advisor; Santangelo, Thomas, committee member; Roess, Deborah, committee member
Menaquinones (MK) are electron carriers composed of a naphthoquinone moiety and an isoprene side chain of variable length and saturation. These molecules are the only quinone derivatives present in the electron transport systems of all Gram-positive bacteria and some Gram-negative anaerobes. Subsequently, MK plays a critical role in respiration for pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis. Although the physiological function and relevance of MK as a redox cofactor have been established, its chemical interactions within the plasma membrane and the effects of these properties on MK-mediated electron transport are still obscure. These unknowns are reflected in existing literature, as MK is commonly depicted in an extended conformation, although in vitro and in vivo studies suggest that biomolecules with alkyl moieties assume folded conformations in native environments (Ko et al., 2011; Trembleau et al., 2003). In this study, we implemented 1D 1H and 2D 1H-1H NMR spectroscopic techniques to characterize the location and 3D conformation of MK-2 within a L-α-phosphatidylcholine liposome model. MK-2, a truncated menaquinone analog, was selected due to its limited rotational variability and previous characterization in a simple monolayer lipid system (Koehn et al., 2018). Our data suggests that MK-2 is largely incorporated into the phospholipid bilayer, with an aqueous subspecies residing at the polar membrane interface in a concentration-dependent manner. 2D NOESY spectroscopic analysis supports the interpretation that both the aqueous form and the membrane-associated form of MK-2 assume a folded conformation. These findings provide a reference for the study of the properties of MK derivatives with longer isoprene chains, which are analogous to functional MK variants in native environments.
Open Access
Characterization of poliovirus 2CATPase bound to bilayer nanodiscs and involvement of the poliovirus 3Dpol thumb α-helix in determining poly(A) tail length
(Colorado State University. Libraries, 2013) Springer, Courtney Lee, author; Peersen, Olve B., advisor; Ho, P. Shing, committee member; Luger, Karolin, committee member; Kennan, Alan, committee member
Poliovirus (PV) is a small non-enveloped picornavirus with a ≈7.5 kb long single-stranded, positive-sense RNA genome. Upon infection, the RNA is translated to generate a ≈250 kDa polyprotein that is subsequently cleaved into about a dozen fully processed proteins and several functional intermediates. PV replication occurs in large membrane associated complexes involving the "non-structural" P2 and P3 region proteins and two of these proteins, 2CATPase and 3Dpol, are the subjects of this dissertation. Part I of this work is focused on the 2C protein, an AAA+ family ATPase that plays a key role in host cell membrane rearrangements and virion assembly, but the membrane binding characteristics of 2C and its polyprotein precursors have made it difficult to elucidate their exact roles in virus replication. In this work I show that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C and the precursor 2BC and 2BC3AB polyproteins in a membrane bound form. Biochemical analysis shows that the proteins are highly active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Notably, the ATPase activity of the core 2C domain is stimulated ≈60-fold as compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. This data leads to a model wherein the viral replication complex can be assembled with a minimally active form of 2C that then becomes fully activated upon proteolytic cleavage from the adjacent 2B viroporin domain. In Part II of this dissertation, I focus on the role of the viral RNA polymerase, 3Dpol, in maintaining the ≈20-150 nucleotides long 3' poly(A) tail of the viral genome. The length of the tail is important for viral replication and initiation of (-)-strand synthesis, but the means by which the RNA is polyadenylated and how poly(A) tail length is regulated is not well understood. We have identified several mutations in an α-helix of the 3Dpol thumb domain that directly impact poly(A) tail length. Here, I tested the impact of these mutations on reiterative transcription of poly(A), poly(U), and poly(C) templates as well as characterized their effect on 3Dpol initiation, stability, elongation rate, and fidelity. I found that mutations in the thumb have the greatest impact on elongation complex stability and that 3Dpol is able to reiteratively transcribe homopolymeric poly(U) and poly(A), but not poly(C) RNA templates. Interestingly, distinct poly(A) and poly(U) transcripts are generated from 10 nucleotide homopolymers that are 1, 7, or 8 nucleotides longer than the template. Based on these findings, we propose a poly(A) slippage model in which the elongation complex stalls at the end of the homopolymer stretch in the absence of additional nucleotides to promote a single nucleotide slippage. This is followed by a slow structural rearrangement in which 3Dpol slips back to the 3' end of the homopolymer sequence, where it is able to re-transcribe starting from the fifth poly(U) in the template.
Open Access
Characterization of the dissociation equilibria of the histone H3H4 tetramer using sedimentation velocity analytical ultracentrifugation
(Colorado State University. Libraries, 2020) Connolly, Mark Edward, author; Hansen, Jeffrey, advisor; Nyborg, Jennifer, committee member; Walrond, John, committee member
By performing sedimentation velocity analytical ultracentrifugation experiments under various ionic and pH conditions the kd can be measured. This system was found to be pH dependent with a change of Svedberg (S) value distribution in between pH 5.2 and pH 5.5. The H3H4 system was found to be an interacting one due to the change in S value distribution with increasing the histone protein concentration. We found that the S value distribution is highly dependent on the ionic conditions of the solution with 2M NaCl solution showing higher S values then 5mM KPO4 solution at the same concentration and pH. Oddly enough adding HEPES to a KPO4 buffer will destabilize the H3H4 species present with 5mM KPO4 having a higher S value distribution than 5mM KPO4 10mM HEPES at the same concentration and pH. I was unable to model these systems to a H3H4 dimer to tetramer equilibrium model which leads me to believe the conditions I was using did not stabilize the tetramer save for the 2M NaCl.
Open Access
Characterization of the selective hydrolysis of branched ubiquitin chains by Uch37 and its activator Rpn13
(Colorado State University. Libraries, 2020) Hazlett, Zachary S., author; Yao, Tingting, advisor; Cohen, Robert, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Kennan, Alan, committee member
The ubiquitin (Ub) C-terminal hydrolase, Uch37, can be found associated with the 26S proteasome as well as the INO80 chromatin remodeling complex. Bound to the 26S proteasome, it assists in regulating the degradation of Ub modified proteins. The proteasomal subunit Rpn13 binds Uch37, anchors it to the proteasome 19S regulatory particle and enhances the deubiquitinating enzyme's (DUB's) catalytic activity. While the structure of the Uch37/Rpn13 complex bound to a single Ub molecule has been characterized, much still remains unknown regarding the enzyme's substrate specificity, the molecular basis for its substrate specificity, and its function in the regulation of proteasomal degradation. In this thesis we characterize the substrate specificity of Uch37 with and without its proteasomal binding partner Rpn13. By synthesizing poly-Ub chains of various linkage types and topologies and using these Ub chains in in vitro deubiquitination assays, we were able to determine that Uch37/Rpn13 selectively cleaves branched Ub chains. This provides evidence to suggest that Uch37 is the first enzyme with activity specific for branched Ub chains. Branched Ub chains have been identified endogenously and have roles connected to the regulation of nascent misfolded polypeptides, cell cycle control, and the enhancement of proteasomal degradation. The work presented here sets out to characterize the molecular mechanism of branched chain hydrolysis by Uch37 and its binding partner Rpn13, determine the kinetics of this enzymatic reaction, and establish a system for probing the function of "debranching" by Uch37 in proteasomal degradation. The conclusion of our work builds our understanding of the complex system of intracellular signaling by Ub and unveils key elements to the primary system responsible for regulating cellular protein homeostasis.
Open Access
Characterizing porous protein crystal materials for applications in nanomedicine and nanobiotechnology
(Colorado State University. Libraries, 2018) Hartje, Luke Fredrick, author; Snow, Christopher D., advisor; Ho, P. Shing, committee member; Peersen, Olve B., committee member; McCullagh, Martin, committee member
Protein crystals are biologically derived, self-assembling, porous structures that have been used for decades in structure determination via X-ray diffraction. Recently, however, there has been increased interest in utilizing protein crystals for their unique material properties—most notably, their highly ordered porous structure, innate biocompatibility, and chemical plasticity. The diverse topologies of protein crystals and the relative ease with which their chemical properties can be altered via genetic mutation or chemical modification offers a wider and more dynamic design palette than existing chemically-synthesized nanoporous frameworks. These traits make protein crystals an attractive new material for applications in nanomedicine and nanobiotechnology. The intent of this project is to demonstrate the application potential of porous protein crystal materials for use in nanostructured devices. This work highlights our efforts to: experimentally and computationally investigate macromolecular transport and interaction energies within a large-pore protein crystal environment using time-lapse confocal microscopy, bulk equilibrium adsorption, and hindered diffusion simulation; assess the cytocompatibility of various cross-linking chemistries for the production of biostable protein crystal materials for use in biologically sensitive environments; and create multifunctional textiles by covalently attaching various cross-linked protein crystals to cellulose fibers in woven cotton fabrics. By pursuing this research, we hope to better understand porous protein crystal materials and leverage that knowledge to design advanced nanostructured devices for applications in medicine and biotechnology.