Theses and Dissertations at Montana State University (MSU)
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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 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 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.