Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
10 results
Filters
Settings
Search Results
Item Mechanisms of gating nucleotide-driven electron transfer in nitrogenase(Montana State University - Bozeman, College of Letters & Science, 2020) Pence, Natasha Kathrine; Chairperson, Graduate Committee: John W. Peters; Monika Tokmina-Lukaszewska, Zhi-Yong Yang, Rhesa N. Ledbetter, Lance C. Seefeldt, Brian B. Bothner and John W. Peters were co-authors of the article, 'Unraveling the interactions of the physiogical reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle' in the journal 'The Journal of Biological Chemistry' which is contained within this dissertation.The Mo-nitrogenase from Azotobacter vinelandii reduces N 2 to ammonia in an ATP-dependent process. It has two-components, the MoFe protein (MoFe) with the active site for N 2 reduction, and the Fe protein (FeP) that delivers electrons to MoFe. The less efficient alternative nitrogenases (Fe- and V-nitrogenases) have FeFe and VFe proteins with an additional subunit, termed gamma, whose role is unknown. Electron delivery to MoFe occurs through the Fe protein cycle (FeP cycle). This involves association between the FeP(MgATP 2) and MoFe, followed by electron transfer, ATP hydrolysis, release of P i, and dissociation of the FeP(MgADP 2) from MoFe. A study of the Fe protein cycle with the physiological electron donor flavodoxin (Fld), changed the rate-limiting step for nitrogenase catalysis, highlighting the important role of physiological protein donors in nitrogenase catalysis. However, it is unknown if Fld interacts with the MgADP or MgATP-bound state of the FeP. Insights from ClusPro 2.0 in silico docking models, time-resolved limited proteolysis and chemical cross-linking coupled with LC-MS and MALDI-TOF MS analysis show that the FeP(MgADP 2) forms a more productive complex with Fld, reducing competition between Fld and MoFe for the FeP(MgATP 2) to drive catalysis. To confirm our model, MicroScale Thermophoresis (MST) was developed to measure binding affinity between the FeP and nucleotides which agreed with previous measurements from isothermal calorimetry, confirming its application for nitrogenase. In silico docking models with ClusPro 2.0 and HADDOCK 2.2 identified structural differences between the Mo-nitrogenase and the alternative V- and Fe-nitrogenases that allow discrimination of protein-protein interactions that enable complex formation. The gamma subunit of the V- and Fe-nitrogenases mediates interactions between the nitrogenases, preventing competition between the least efficient Fe-nitrogenase and the Mo-nitrogenase. Finally, a pipeline was developed for homology modeling of potential physiological donor ferredoxin proteins (VnfF, FdxN, FixFd) associated with expression of the Mo-, V- or Fe-nitrogenases. Insights from in silico docking and assessment with the PRODIGY server were used to identify structural features that differentiate how these ferredoxins interact with the FePs of the three nitrogenases. Ultimately, nucleotide-dependent control of protein-protein interactions is necessary to support N 2 reduction and funnel electrons to the most efficient Mo-nitrogenase.Item Fluorescence quenching in 2-aminopurine-labeled model DNA systems(Montana State University - Bozeman, College of Letters & Science, 2019) Remington, Jacob Michael; Chairperson, Graduate Committee: Patrik R. Callis; Abbey M. Philip, Mahesh Hariharan and Bern Kohler were co-authors of the article, 'On the origin of multiexponential fluorescence decays from 2-aminopurine labeled dinucleotides' in the journal 'The journal of chemical physics' which is contained within this thesis.; Martin McCullagh and Bern Kohler were co-authors of the article, 'Molecular dynamics simulations of 2-aminopurine-labeled dinucleoside monophosphates reveal multiscale stacking kinetics' in the journal 'Journal of physical chemistry B' which is contained within this thesis.For the last 50 years changes to the fluorescence properties of 2-aminopurine have been used to probe the structure and dynamics of DNA. 2-Aminopurine's utility has arisen from the quenching of its emission when pi-stacked with neighboring nucleobases. In the time-domain, the emission decay profile of 2-aminopurine requires multiple exponential decay components to model. Despite its extensive usage, the microscopic origin of the decay heterogeneity is not clear. In this thesis, steady-state absorption, fluorescence, and time-resolved fluorescence results are compared to multiple microsecond molecular dynamics simulations of 2-aminopurine-labeled adenine containing single-stranded DNA oligomers of varying length and position of the 2-aminopurine probe. First, previous reports of ultrafast electron transfer in pi-stacked adenine oligomers are used to build a new model for quenching of 2-aminopurine that is pi-stacked with adenine. For dinucleotides, a static distribution of unstacked structures combined with a distance dependent electron transfer mechanism is posited to explain the disperse emission decay timescales. Investigating the dinucleotides with molecular dynamics simulations analyzed with Markov state models quantify the structural heterogeneity of the dinucleotides. At least seven structures are sampled that could alter the quenching of 2-aminopurines's fluorescence. The Markov state models also demonstrate the timescales for transitions between these structures range from 1.6 to 25 ns, suggesting 2-aminopurine, with its monomer-like lifetime of 10 ns, is sensitive to the conformational dynamics of the dinucleotides as well. This dual fluorescence quenching and molecular dynamics simulation approach is extended to 2-aminopurine labeled trinucleotides and 15 base oligomers to interrogate the position dependent structural heterogeneity and conformational dynamics in these systems. Both shifts in the experimental absorption spectra, and molecular dynamics simulations agree that the interior base is more likely to be stacked than the exterior bases. Time-resolved emission experiments reveal emission from 2-aminopurine is quenched faster on the 5' end relative to the 3' end, in agreement with the faster stacking kinetics observed for bases on the 5' end relative to the 3' end obtained from molecular dynamics simulation. These results suggest that the time-resolved emission from 2-aminopurine may serve as an experimental observable for calibration of the dynamical properties predicted by molecular dynamics simulation.Item Transduction of antigens into amplifiable DNA signals using structure switching aptamers(Montana State University - Bozeman, College of Engineering, 2019) Kayalar, Canberk; Chairperson, Graduate Committee: Stephanie McCallaDetection of specific antigens has one vital step in common: detection of biomarkers. Diagnostic testing that is rapid and reliable is unavailable in limited resource and rural settings. The solution to this need must be simple, inexpensive, robust, rapid and not require highly trained personnel to operate. Aptamers are capable of delivering those needs when matched with a novel high gain amplification method. This thesis focuses on important aspects of a novel protein detection assay that uses aptamers. Aspects that play an important role on the assay's success were investigated; aptamer selection and design of structure switching aptamers, designing DNA templates that will transduce the signal created by the aptamers, solid phase selection, aptamer immobilization on the solid phase, protein capture, and amplification of the signal. The first step was to find aptamers that were proven to specifically target clinically relevant targets and modify them to suit the needs of the assay. It is important to validate the aptamers' performance. The second important step was finding a solid phase that is compatible with the novel nucleotide amplification reaction that will be used to amplify the signal produced by the aptamers. Paramagnetic microbeads, membranes and polyacrylamide hydrogels were potential candidates for solid phases. Non-specific interaction of the target protein with the solid phase surface will not have negative effects while running the assay due to the structure switching of the aptamers however, it prevented the accurate quantification of the protein capture by aptamers. There is a need for the development of a blocking buffer that is specific to the solid phase. Washing of the excess DNA templates that are not bound to target-bound aptamers plays an important role in the assay's accuracy. The results presented here show the preliminary work that has been done for the novel protein detection assay that uses structure switching aptamers. This assay has the potential to detect diseases at point-of-care in low resource settings.Item A mathematical model of a biphasic DNA amplification reaction(Montana State University - Bozeman, College of Letters & Science, 2019) Ciesielski, Danielle Kristine; Chairperson, Graduate Committee: Tomas GedeonIsothermal DNA amplification reactions have many applications ranging from analyte detection to DNA circuits. EXPonential Amplification Reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This Ultrasensitive DNA Amplification Reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms. We show that the model can reproduce the experimentally observed biphasic behavior. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding caused by two palindromic trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. Understanding of these mechanisms also illuminates behavior of EXPAR and other nucleic acid amplification reactions. For a deeper understanding of the roles these mechanisms play in DNA amplification reactions, we apply dynamical systems analysis to the model. We first consider the long term behavior of partial models that lack key reaction mechanisms described above to see how their omission impacts the system's overall behavior. Then we use perturbation theory to examine the time scales on which these mechanisms operate and how their interaction leads to biphasic growth. We find that mechanisms (i) and (ii) together create a stable equilibrium reminiscent of EXPAR reactions, but the addition of mechanism (iii) changes the stability of this equilibrium and generates UDAR's characteristic high amplification. Finally, mechanism (iv) introduces a second stable equilibrium that indicates that polymerase sequestration is the mechanism that ends the second fast amplification phase. In addition, throughout this work we identify which rate constants shape different parts of the biphasic growth. These results can guide future work in rational design of molecular detection assays.Item Validating Salmonella typhimurium virulence modulation by ecto-5'-nucleotidase (CD73) in intestinal epithelial cells(Montana State University - Bozeman, College of Letters & Science, 2017) Schneider, Stephanie Lorain; Chairperson, Graduate Committee: Douglas KominskyExtracellular ATP is a pro-inflammatory molecule released during intestinal insult and must be converted to adenosine by ecto-5'-nucleotidase (CD73) for the resolution of intestinal inflammation [1]. Along with its anti-inflammatory role in the intestinal mucosa, CD73-generated adenosine contributes to host-microbe interactions at the mucosal surface by modulating pathogen replication and virulence, including that of Salmonella enterica serovar Typhimurium (S. Typhimurium) [2], [3]. It has been shown, in the absence of intestinal epithelial cell-specific CD73, S. Typhimurium virulence is attenuated in vivo and in vitro, implicating the intestinal epithelium as an underappreciated source for the development of novel antimicrobial therapies. Since direct modulation of extracellular adenosine leads to pleiotropic effects, the aim of this research was to determine the mechanism(s) of S. Typhimurium virulence modulation by CD73 in intestinal epithelial cells to identify specific molecular targets that modulate pathogenesis [4], [5]Item Theory-based demarcation of hot spring microbial mat species from large DNA sequence datasets(Montana State University - Bozeman, College of Agriculture, 2018) Wood, Jason Michael; Chairperson, Graduate Committee: David M. Ward; Eric D. Becraft, Danny Krizanc, Frederick M. Cohan, and David M. Ward were co-authors of the article, 'Ecotype simulation 2: an improved algorithm for efficiently demarcating microbial species from large sequence datasets' submitted to the journal 'BMC bioinformatics' which is contained within this thesis.; Jason M. Wood, Frederick M. Cohan and David M. Ward were co-authors of the article, 'Biogeography of American Northwest Hot Spring A/B'-lineage Synechococcus populations' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.The identification of closely related, ecologically distinct populations within microbial communities is paramount to understanding the structure and function of these communities. Microbial systematists have long used differences in DNA sequence relatedness to categorize the observed diversity in a community of microbes without including ecological theory to identify whether or not the identified groups are ecologically distinct. Ecotype Simulation, an evolutionary simulation algorithm based on the Stable Ecotype Model of microbial species and speciation, has been used successfully to study the diversification of thermophilic A/B'-lineage Synechococcus living in the effluent channels of alkaline-siliceous hot springs in Yellowstone National Park. However, Ecotype Simulation is an extremely slow program that is unable to handle the quantity of data produced by modern DNA sequencing technologies. I introduce a new version of this algorithm, called Ecotype Simulation 2, that permits the rapid analyses of microbial diversity from very large DNA sequence datasets. Results from this new version of the Ecotype Simulation algorithm compare favorably with results from the old version, but with analyses performed much more quickly on a much greater quantity of sequences sampled. The new algorithm was used to analyze three datasets. First, the biogeography of thermophilic A/B0-lineage Synechococcus living in hot springs of the American Northwest was analyzed. Results suggested a surprising amount of endemism among springs sampled, as well as implications for adaptations to physical and chemical environmental features not seen before. Second, Ecotype Simulation 2 was used to study the history of change in Synechococcus populations, seasonally (winter to summer) and over a twenty-five year period. Results suggested changes in population abundances and distribution seasonally, but stability in population genetic structure over many years. Finally, Ecotype Simulation 2 was used to study the populations of other predominant phototrophic microbes living along temperature and depth gradients in the same microbial mat community. Results suggested that the algorithm and the Stable Ecotype Model can successfully predict ecological diversity within all predominant mat taxa. Ecotype Simulation 2 provides the means for other microbiologists to base their understanding of the communities they study on evolutionary and ecological principals.Item Photochemistry and photophysics of guanines(Montana State University - Bozeman, College of Letters & Science, 1974) Morgan, James PaulItem Polarized fluorescence of the dinucleotides(Montana State University - Bozeman, College of Letters & Science, 1974) Wilson, Robert WilfredItem Identificaton of microsatellite markers associated to a solid stem QTL in wheat(Montana State University - Bozeman, College of Agriculture, 2003) Cook, Jason PatrickItem Nucleotide dependent conformational changes in the nitrogenase Fe protein(Montana State University - Bozeman, College of Letters & Science, 2005) Sen, Sanchayita; Chairperson, Graduate Committee: John W. PetersNitrogenase is a complex metal-containing enzyme that catalyzes the conversion of nitrogen gas to ammonia. During nitrogenase catalysis the Fe protein and the molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. The role of nucleotide binding and hydrolysis in nitrogenase catalysis is one of the most fascinating aspects of nitrogenase function. The Fe protein upon binding to MgATP undergoes a huge conformational change which is important for subsequent steps of nitrogenase reaction mechanism. Therefore structural characterization of the Fe protein bound to MgATP will provide a basis on how MgATP binding promotes the complex formation whereas hydrolysis to MgADP leads to the dissociation of the macromolecular complex structure. Towards these ends we have conducted structural studies on a site-directed variant of the Fe protein which is a close mimic of the MgATP conformational state. Structural characterization of this Leu127 deletion variant revealed a distinctly new conformation of the Fe protein which arises from the rigid body reorientation of the homodimeric Fe protein subunits with respect to each other. The structure not only provides the first basis on rationalizing the initial docking interactions between the component proteins but also helps us to dissect the conformational changes on the Fe protein which occur upon nucleotide binding from those conformational changes that are imposed on the Fe protein by the MoFe protein during complex formation. Having this structure in hand, we have developed several other experimental approaches like Mass spectrophotometry and Small Angle X-ray Scattering/Diffraction (SAXS) techniques to probe the relationship between the Leu127 deletion variant a close structural mimic of MgATP bound "on state" and the actual MgATP bound state which is more difficult to probe crystallographically. These studies will help us to compare the different nucleotide bound states (MgADP and MgATP) of the Fe protein in solution that will help to predict the level of conformational change that is induced in the Fe protein that makes it compatible for binding to the MoFe protein in the nitrogen catalysis cycle.