Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
Browse
9 results
Filters
Settings
Search Results
Item Allosteric activation of the CRISPR-associated transcription factor Csa 3 by cyclic tetra-adenylate (cA 4)(Montana State University - Bozeman, College of Letters & Science, 2020) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin LawrenceThe CRISPR-Cas immune system provides adaptive and heritable immunity to archaea and bacteria to combat viral infection, and is a source of biochemical tools to researchers. This work combines structural biology and biochemical approaches to provide insight into mechanisms prokaryotes use to control the CRISPR-Cas immune system, linking subsystems into a coordinated response. The first structure of S. solfataricus Csa3 determined by Lintner et al. revealed a dimer with a C-terminal wHTH DNA-binding domain and an N-terminal CARF domain with a putative ligand binding site predicted to bind a two-fold symmetric molecule with both negatively charged and hydrophobic/aromatic moieties, such as dinucleoside polyphosphates or nucleic acid molecules. 1 Later, analysis by Topuzlu et al. of the A. fulgidis Csx3 structure containing a 4 base RNA molecule in a binding pocket revealed similarities between Csx3 and the Csa3 CARF domain, and suggested CARF proteins could bind cyclic or pseudosymmetric linear RNA tetranucleotides represented by the ring-shaped RNA density in the Csx3 binding pocket. 2 Functional studies with S. islandicus Csa3 identified that SiCsa3 regulates transcription of acquisition genes (cas1, cas2, and cas4) and several CRISPR loci. 3,4 Additionally, two groups simultaneously showed that the Type III surveillance complexes produce cyclic oligoadenylate messengers, including cyclic tetra-adenylate (cA 4), which allosterically regulate the RNase activity of Csm6 and Csx1, other CARF proteins. 5,6 These advances support the original predictions by Lintner et al. and suggest that Csa3 binds a cyclic RNA as proposed by Topuzlu et al. Binding of cA 4 likely allosterically causes conformation changes in the wHTH, and regulates the protein's transcriptional regulation. We present the crystal structure of S. solfataricus Csa3 complexed with cyclic tetraadenylate (cA 4). cA 4, as predicted, 1 is bound in the CARF domain 2-fold symmetric pocket, which stimulates conformational changes in the C-terminal domain. Additionally, we identify the presence of a palindromic predicted binding motif upstream of the Type I-A(2) acquisition cassette and CRISPR loci C and D, and reveal through EMSA analysis that Csa3 binds dsDNA nonspecifically with high affinity. Finally, ring nuclease activity is not detected in Csa3, suggesting longer term potentiation of the cA 4 in Csa3 than observed for Csx1/Csm6. 5,6Item 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 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 The emergence of collective behavior on social and biological networks(Montana State University - Bozeman, College of Letters & Science, 2018) Wilander, Adam Troy Charles; Chairperson, Graduate Committee: Scott McCallla; Dissertation contains an article of which Adam Troy Charles Wilander is not the main author.In this thesis, we broadly examine collective behaviors in various social and biological contexts. Aggregation, for instance, is a natural phenomenon that occurs in a variety of contexts; it is observed in schools of fish, flocks of birds, and colonies of bacteria, among others. This behavior can be found in some agent-based models, where it is typically assumed every pair of individuals interact according to a simple set of rules. In the first half of this thesis, we study a particular, well-understood aggregation model upon relaxation of the assumption that every individual interacts with every other. We review prior results on this topic -- when the underlying structure of interactions is an Erdos-Renyi graph. Seeking to incorporate community structure into the network, we establish the analogous problem under a class of networks called stochastic block graphs; a particular aspect of the system's metastable dynamics is explored upon varying the graph's connection densities. Finally, we evaluate the potential to leverage this system's dynamics in order to recover community structure (given a known graph as input). In the second half of this thesis, we similarly explore the aggregate behaviors of synchronization and desynchronization, appearing in diverse settings such as the study of metabolic oscillations and cell behaviors over time, respectively. Previous studies have leveraged a model in which repressilator entities are connected by a diffusive quorum sensing mechanism; these have shown (numerically) that the complex composition of observable behaviors depends upon the insertion point of the upregulating protein in the feedback loop. We rigorously prove a version of this; for negative feedback, negative signaling (Nf-Ns) systems we find only a unique stable equilibrium or a stable oscillation is possible. Additionally, we observe (numerically) the complex multistable dynamics that arise when a positive signal is included in the feedback loop and characterize this shift as a saddle node bifurcation of a cubic curve.Item Investigation of a control strategy for manipulation and prevention of Pseudomonas aeruginosa PAO1 biofilms in metalworking fluids(Montana State University - Bozeman, College of Engineering, 2018) Ozcan, Safiye Selen; Chairperson, Graduate Committee: Christine Foreman; Markus Dieser, Albert E. Parker, Narayanaganesh Balasubramanian and Christine M. Foreman were co-authors of the article, 'Quorum sensing inhibition as a promissing method to control biofilm growth in metalworking fluids' submitted to the journal 'Environmental science & technology' which is contained within this thesis.Microbial contamination in metalworking fluid (MWF) circulation systems is a serious problem. Particularly water based MWFs promote microbial colonization despite the use of biocides. Inhibiting the quorum sensing mechanism (i.e. cell-cell communication) in bacteria is a promising approach to control and prevent biofilm formation. The objective of this study was (i) to determine the microbial community in MWFs from operational machining shops, (ii) to investigate the effect of well-known quorum sensing inhibitors on controlling biofilm formation, and (iii) to implement experimental data from selected enzymes to a computer simulation biofilm accumulation model (BAM). Planktonic and biofilm samples from two local machining shops in Bozeman, MT, were collected to determine the extent of microbial colonization. In both operations, microbial communities were dominated by Pseudomonadales (60.2-99.7%). Rapid recolonization was observed even after dumping spent MWFs and cleaning. Considering the dominance of Pseudomonadales in MWFs, the model organism Pseudomonas aeruginosa PAO1 was selected for testing the effects of quorum sensing inhibitor compounds on biofilm formation. From a variety of enzymes, natural, and chemical compounds screened for quorum sensing inhibition, Patulin (40microns) and Furanone C-30 (75microns), were found to be effective in reducing biofilm formation in MWFs when applied as single compound amendments and in combination with the polysaccharide degrading enzyme alpha-amylase from Bacillus amyloliquefaciens. Particularly Furanone C-30, as a single amendment and in combination with alpha-amylase decreased biofilm formation by 76% and 82% after 48 hours. Putatively identified homoserine lactones in MWFs treated with Furanone C-30 provided evidence for quorum sensing inhibition on biofilm formation. BAM was employed to study the effect of alpha-amylase (3 Units mL -1) on P. aeruginosa PAO1 biofilms in batch reactors for 24 and 48 hours. In the absence of alpha-amylase, biofilm thickness was predicted to be 23.11 and 31.37 microns, while its presence reduced thickness to 10.47 and 13.07 microns after 24 and 48 hours, respectively. The results presented herein highlight the potential effectiveness of quorum sensing inhibition as a strategy to reduce biofilms in MWFs.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 Developmental differences in children in hand temperature control through biofeedback(Montana State University - Bozeman, College of Letters & Science, 1988) Georgitis, Jane GrazierItem Effects of various coping strategies (Lamaze vs. biofeedback vs. telling) on relaxation as measured by EMG and finger-tip temperature in nine primiparas(Montana State University - Bozeman, College of Nursing, 1979) Caldwell, Margo MarieItem The effects of autogenic exercises and the ability of college students to think abstractly as measured by electromyographic biofeedback(Montana State University - Bozeman, College of Education, Health & Human Development, 1982) Weems, Dennis Lorry