Theses and Dissertations at Montana State University (MSU)
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Item Investigating the role of allostery through changes in protein stability and dynamics(Montana State University - Bozeman, College of Letters & Science, 2021) Patterson, Angela Jean; Chairperson, Graduate Committee: Brian Bothner; Faiz Ahmad and Jaigeeth Deveryshetty were authors and Jenna R. Mattice, Nilisha Pokhrel, Brian Bothner and Edwin Antony were co-authors of the article, 'Hydrogen-deuterium exchange reveals a dynamic DNA-binding map of replication protein A' in the journal 'Nucleic Acids Research' which is contained within this dissertation.; Zhongchao Zhao, Elizabeth Waymire, Adam Zlotnick, and Brian Bothner were co-authors of the article, 'Dynamics of hepatitis B virus capsid protein dimer regulates assembly through an allosteric network' in the journal 'ACS chemical biology' which is contained within this dissertation.; Paul B.G. van Erp was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and Cas 3 recruitment by the CRISPR RNA-guide cascade complex' in the journal 'ACS chemical biology' which is contained within this dissertation.; Aidan White, Elizabeth Waymire, Sophie Fleck, Sarah Golden, Royce Wilkinson, Blake Wiedenheft and Brian Bothner were co-authors of the article, 'Thermodynamics of CRISPR-anti-CRISPR interactions provides mechanistic insight into inhibition' which is contained within this dissertation.Allostery is the presence of a communication network that links functional sites of a protein that are distal from one another. The existence of an allosteric network can be observed through conformational change or a change in protein dynamics. These networks can be used to provide insight into the mechanistic function of proteins or protein complexes. In this thesis, four protein complexes were studied (RPA, HBV, Cascade, and Csy) and allosteric networks within the complexes were observed by monitoring the changes in protein dynamics upon an energy perturbation. To measure the changes in protein dynamics, hydrogen deuterium exchange mass spectrometry was used. This technique allows for the determination of how often the hydrogen bonding within a protein structure is broken. By tracking the longevity of the hydrogen bonding network that comprises the studied protein's structure, the dynamics of the protein can be studied. In this work, each of the proteins had changes in protein dynamics that were distal from the site of the energy perturbation that had functional impacts on each of the protein complexes. The combined presence of the distal changes in dynamics with an effect on protein function fits the definition of allostery. If allostery is present in these four diverse systems, is it possible that allostery is present in all proteins?Item Morphological adaptations facilitating attachment for archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2019) Hartman, Ross Alan; Chairperson, Graduate Committee: Mark J. YoungLittle is known regarding the attachment and entry process for any archaeal virus. The virus capsid serves multiple biological functions including: to protect the viral genome during transit between host cells, and to facilitate attachment and entry of the viral genome to a new host cell. Virus attachment is conducted without expenditure of stored chemical energy i.e. ATP hydrolysis. Instead, virus particles depend on diffusion for transportation and attachment from one host cell to another. This thesis examines the attachment process for two archaeal viruses. Sulfolobus turreted icosahedral virus (STIV) is well characterized for an archaeal virus. Still, no information is available concerning STIV attachment or entry. The research presented here shows that STIV attaches to a host cell pilus. Furthermore, combining the previously determined atomic model for the virus, with cryo-electron tomography, a pseudo-atomic model of the interaction between the host pilus and virus was determined. Based on this data, a model is proposed for the maturation of the virus capsid from a noninfectious to an infectious form, by dissociation of accessory proteins. Finally, a locus of genes is identified in the host cell, encoding proteins essential for viral infection, that are likely components of the pili structure recognized by STIV. The isolation of a new archaeal virus, Thermoproteus Piliferous Virus 1 (TSPV1), is also presented here. The TSPV1 virion has numerous fibrous extensions from the capsid, of varying length, that are the first observed for any virus. The capsid 2-3nm fibers likely serve to extend the effective surface area of the virus, facilitating attachment to host cells. Characterization of this new virus was conducted, including genome sequencing and determination of the protein identity for the capsid fibers. The research presented here provides a substantial advancement in our knowledge of the attachment process for archaeal viruses. Attachment to host pili is now emerging as a common theme for archaeal viruses. Furthermore, the isolation of the new archaeal virus TSPV1 demonstrates a novel strategy to increase the probability of interaction between a virus and host cell.Item Relating protein structure to function: how protein dynamics maximizes energy gained by electron transfer in an anaerobic energy conservation mechanism(Montana State University - Bozeman, College of Letters & Science, 2019) Berry, Luke Montgomery; Chairperson, Graduate Committee: Brian Bothner; Angela Patterson, Natasha Pence, John Peters and Brian Bothner were co-authors of the article, 'Hydrogen deuterium exchange mass spectrometry of oxygen sensitive proteins' in the journal 'Bio-protocols' which is contained within this dissertation.; Saroj Poudel, Monika Tokmina-Lukaszewska, Daniel R. Colman, Diep M.N. Nguyen, Gerrit J. Schut, Micheal W.W. Adams, John W. Peters, Eric S. Boyd and Brian Bothner were co-authors of the article, 'H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle' in the journal 'Biochimica et biophysica acta (BBA) - general subjects' which is contained within this dissertation.; Monika Tokmina-Lukaszewska, Derek F. Harris, Oleg A. Zadvornyy, Simone Raugei, John W. Peters, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Combining in-solution and computational methods to characterize the structure-function relationship of the nitrogengase systems' which is contained within this dissertation.; Hayden Kallas, Derek F. Harris, Monika Tokmina-Lukaszewska, Simone Raugei, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Iron protein docking effects on MOFE protein dynamics: function of negative cooperativity and the regulation of electron trasfer' which is contained within this dissertation.Reduced ferredoxin (Fd) plays a critical role in anaerobic metabolism by acting as an alternative source of energy to adenosine triphosphate (ATP). The reduction potential of Fd is low (-450 mV) making it difficult to reduce individually. However, it has recently been discovered that a unique mechanism known as electron bifurcation allows anaerobic organisms to reduce Fd without suffering a loss of energy. Electron bifurcation was originally discovered in complex III of the electron transport chain, and increased the efficiency of the proton motive force without an overall change in the electron flow, minimizing energy loss. EB accomplishes this is by coupling a favorable (exergonic) and unfavorable (endergonic) reduction reaction. The exergonic reaction produces a singly reduced cofactor with a sufficiently negative reduction potential to allow the endergonic process to proceed. This allows anaerobic organisms to couple the formation of NADH, with the reduction of Fd. A detail of interest in the bifurcating mechanism is how these enzymes regulate the flow of electrons down the exergonic and endergonic branches to prevent multiple electrons from traveling down the exergonic branch. It is hypothesized that changes in the protein conformation alter the distance between cofactors altering the rate of electron transfer. To fully understand how changes in a protein's conformation regulates electron transfer in electron bifurcation we used a suite of in-solution techniques, such as H/D exchange and chemical cross-linking coupled to mass spectrometry to characterize the structure and dynamics of the model bifurcating enzyme, NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn), during the different steps of electron bifurcation. Additionally we also set out to use these techniques to characterize the structure and dynamics of the nitrogenase systems in order to obtain biophysical evidence of negative cooperativity in the various nitrogenase systems.Item Multivalently presented carbohydrates can be used as drug delivery vehicles and to study protein carbohydrate interactions(Montana State University - Bozeman, College of Letters & Science, 2018) VanKoten, Harrison Wesley; Chairperson, Graduate Committee: Mary J. Cloninger; Wendy M. Dlakic, Robert Engel and Mary J. Cloninger were co-authors of the article, 'Synthesis and biological activity of highly cationic dendrimer antibiotics' in the journal 'Molecular pharmaceutics' which is contained within this thesis.; Rebecca Moore, Coleen Murphy and Mary J. Cloninger were co-authors of the article, 'Probing the LEC-1 and LEC-10 oxidative stress pathway in Caenorhabditis elegans using GALBeta1-4FUC dendrimers' which is contained within this thesis.Dendrimers in general excel as drug delivery vehicles since there are many different ways they can be assembled and different ways to tailor them to the system being studied. Glycodendrimers are generally nontoxic and can be further developed to meet the needs of what is being studied. For instance, in the studies below, a quaternity ammonium compound (QAC) has been attached to a glycodendrimer to determine the antimicrobial activity of a multivalently presented QAC in studies of minimum inhibitory concentration (MIC), biofilm prevention, and bacterial resistance. Results include comparable MICs to those of established antibiotics, prevention of biofilm formation but not disruption of an established biofilm, and establishment of multivalency as a strategy to counteract bacterial resistance. Another heterogeneously functionalized dendrimer was synthesized to study drug release characteristics of a prodrug attached to a cleavable substrate. In these studies, the upregulation of several proteins during cancer progression was taken advantage of including; MMP-2, -7, -9, and galectin-3. Glycodendrimers are tools used to study protein carbohydrate interactions. Study of galectins and their corresponding Beta-galactosides have illuminated their role in several essential biological processes. Multivalency plays a crucial role in many protein-carbohydrate interactions. Galectins are known to interact multivalently with various ligands. Although the role of galectins in this process is not yet fully understood, galectins have been proposed to serve as protective proteins during periods of high oxidative stress. We describe the synthesis of GalBeta1-4Fuc functionalized poly(amidoamine) (PAMAM) dendrimers in order to test C. elegans' response to high oxidative stress. In order to test the function of GalBeta1-4Fuc in vivo, C. elegans were treated with RNAi to knockdown lec-1 or lec-10, and then treated with glycodendrimer and exposed to oxidative stress. C. elegans that were pre-treated with the glycodendrimers were less susceptible to oxidative stress than untreated controls. The glycodendrimers mainly appeared within the digestive tract of the worms, and uptake into the vulva and proximal gonads could also be observed in some instances. This study indicates that multivalently presented GalBeta1-4Fuc can protect C. elegans from oxidative stress by binding to galectins.Item Partitioning of reactive oxygen species via the re-oxidation of electron transfer flavoprotein(Montana State University - Bozeman, College of Letters & Science, 2019) Austvold, Chase Kennor; Chairperson, Graduate Committee: Edward DratzThe biology of Reactive Oxygen Species are poorly understood. Within a healthy cell, Reactive Oxygen Species behave as signaling molecules, although overproduction leads to oxidative damage. In order to understand when the overproduction of Reactive Oxygen Species takes place, or leads to oxidative damage, the elementary step of quantification becomes necessary. Electron Transfer Flavoprotein is a known Reactive Oxygen Species producing enzyme and was studied. Electron Transfer Flavoprotein is a key-player within the production of energy within the eukaryotic mitochondria. The redox nature of Electron Transfer Flavoprotein's catalytic cofactor, flavin adenine dinucleotide produced two types of ROS; the superoxide anion (O 2 °-) and hydrogen peroxide (H 2 O 2). Electron Transfer Flavoprotein produced roughly five-fold more O 2 °-compared to H 2 O 2 as the enzyme became oxidized. It has been put forward that the production of these two Reactive Oxygen Species is dictated by the formation of a radical pair between the flavin adenine dinucleotide of Electron Transfer Flavoprotein and molecular oxygen. Two types of radical pairs can be formed, either in a triplet or singlet state, and the rate in which these states occur can be influenced by a static magnetic field. Therefore, the effect of a magnetic field on these products was also studied. Upon the suppression of magnetic field strength, the production of H 2 O 2 decreased and a proportional increase of O 2 °-was observed.Item Development of a protein-based sensor assay for rapid classification of complex biological samples(Montana State University - Bozeman, College of Letters & Science, 2016) Hamerly, Timothy Kyle; Chairperson, Graduate Committee: Brian Bothner; Joshua Heinemann, Monika Tokmina-Lukaszewska, Elizabeth R. Lusczek, Kristine E. Mulier, Greg J. Beilman and Brian Bothner were co-authors of the article, 'Bovine serum albumin as a molecular sensor for the discrimmination of complex metabolite samples' in the journal 'Analytica chemica acta' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Adding metrics to the aging of whiskey using a protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Analysis of wine using the protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Investigations into the use of a protein sensor assay for metabolite analysis' in the journal 'Applied biochemistry and biotechnology' which is contained within this dissertation.Metabolomics, one of the core 'omics' fields within the umbrella of systems biology, is the study of the small molecules which can be used to characterize the state of an organism. Metabolites are constantly being transformed inside a cell in direct response to stimuli around them. This makes the metabolome the most dynamic of all the omics fields and is considered to be a direct readout of the cells state at any given time. Although highly informative, the metabolome is inherently difficult to study, with thousands of known metabolites, any of which could be important for classifying a cell into a healthy or diseased state. Techniques such as mass spectrometry are well suited to study the metabolome and have been used to successfully classify cells by identify markers for a given disease state. However, current methods require lengthy analysis times due in part to the complexity of the metabolome. The research presented in this dissertation highlights a new and promising methodology which improves classification and speeds marker discovery. Making use of a protein found in animals which has evolved to selectively bind metabolites, an assay was developed which better classified samples compared to current methods used in the field of metabolomics. This improved classification was achieved with an overall decrease in analysis time. The implementation of this method in the study of complex biological systems would have an immediate impact in academic and medical research.Item Analysis of conformational dynamics in Hepatitis B capsid protein(Montana State University - Bozeman, College of Letters & Science, 2015) Movahed, Navid; Chairperson, Graduate Committee: Valerie Copie; Brian Bothner (co-chair)Hepatitis B virus (HBV) is a model system for investigating the principles of icosahedral capsid assembly and a major human pathogen. As detailed by the work presented herein, viral capsids are not simply a static container for the viral genome. Rather, they are highly functional molecular machines critical to the virus life cycle. The assembly process of the HBV capsid involves the concerted assembly of 120 homodimeric subunits to form a T=4 icosahedron, which has been shown to be affected by temperature, ionic strength, and small molecules in a manner consistent with models of allosteric regulation. Our lab has already completed rigorous measurements of the conformational equilibria for HBV protein using enzyme-mediated kinetic hydrolysis, where we investigated the role of potential molecular switches in capsid assembly. These studies have now been complemented with hydrogen deuterium exchange based mass spectrometry. Hydrogen deuterium exchange mass spectrometry (HDX-MS) provides valuable insight into solution-phase protein conformation and structure. The resolution of protein structural information in HDX-MS measurements is primarily limited by the peptide coverage of the on-line pepsin proteolysis. We have realized near single amino acid resolution coverage maps by combining online proteolysis with rapid reverse-phase chromatography of highly rich peptide mixtures. Through the use of differential HDX, I investigated the effect of temperature, salt and amino acid mutation on rate of uptake and protection. These effectors have proven to thermodynamically or/and kinetically target the capsid assembly. High resolution HDX-MS was used to investigate the effect of these effectors on the protein dynamics. This has allowed us to elucidate the allosteric mechanism involved in the capsid assembly. Together these results indicate that the conformational landscape of HBV can be remodeled by a range of factors. The ability to map protein motions by HDX on specifically selected conformational states has profound implications in revealing quasi-equivalent subunit associations and the design of antiviral therapies.Item Preparation of egg albumin solutions and observations on their optical activity during heat denaturation(Montana State University - Bozeman, College of Letters & Science, 1950) Cascaden, Norman D.Item Studies of the action of molecular singlet oxygen on proteins and amino acids(Montana State University - Bozeman, College of Letters & Science, 1974) Fischer, James RossItem The effects of proteins from skim milk and other sources on tomatoes(Montana State University - Bozeman, College of Letters & Science, 1951) Dawson, Frederick C.
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