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Item The role of fixX in electron bifurcation(Montana State University - Bozeman, College of Letters & Science, 2016) Miller, Jacquelyn Marie; Chairperson, Graduate Committee: John W. PetersTwo known methods of physiological energy conservation are substrate level phosphorylation and electron transfer phosphorylation. Recently, electron bifurcation has been established as a third and key mechanism of energy conservation in biological processes. This coupling of endergonic and exergonic reactions allows for utilization of reducing potential to perform energetically expensive physiological reactions. A significant and energetically expensive physiological reaction is nitrogen fixation, which provides a substantial portion of the bioavailable nitrogen that life requires. Electron bifurcation is utilized by the FixABCX system that is up regulated during diazotrophic growth and is suggested to bifurcate electrons from NADH to quinone of the electron transport chain through high potential electron transfer proteins and to nitrogenase though low potential electron transfer proteins. The determination of how cellular mechanisms overcome the energy barriers of high potential electron transfers through electron bifurcation is crucial for our fundamental understanding of energy transfer and energy conservation. The work presented in this thesis aims to progress the present knowledge in this third mechanism of energy conservation and shows support for a protein in the FixABCX complex, FixX, as the low potential electron acceptor in the complex. Numerous organisms were investigated as potential model systems for FixABCX with varying degrees of success. The genome of the organism, Roseiflexus castenholzii, contains both the nitrogenase and fixABCX genes and has successfully been used to obtain FixX. This protein shows homology to ferredoxin, a physiological reductant of the nitrogenase Fe protein in some organisms. EPR spectroscopy and sequence analysis suggests FixX contains 2 [4Fe-4S] clusters, while a potentiometric titration shows the clusters to have highly negative mid-point potentials. The preliminary evidence supports FixX of the FixABCX system to be a low potential electron transfer protein.Item Ion transport by viscous gas flow through capillaries and electrospray mass spectrometry of proteins(Montana State University - Bozeman, College of Letters & Science, 1992) Lin, BaiItem Quantitative prediction of dye fluorescence quantum yields in proteins(Montana State University - Bozeman, College of Letters & Science, 2009) Hutcheson, Ryan Mitchell; Chairperson, Graduate Committee: Patrik R. CallisThe application of a method previously developed by Callis et al. to predict the quantum yields of Trp fluorescence has been successfully applied to the fluorescence of fluorescein and flavins in proteins. The calculated lifetime range of 2 ps - 4 ns is in agreement with experiment. The fluctuations in the electron transfer rate are shown to be dictated by the fluctuations in the density of states. This is evident by the comparison of the fractional deviation of the interaction, density of states and the rate. Here the fluctuations in the density of states is an order of magnitude larger than the fluctuations in the interactions and is nearly the same as that of the kET fluctuations. This demonstrates that the fluorescence lifetime variability is controlled by the electrostatic environment and not the distance dependence of the interaction.Item Using PAMAM dendrimer frameworks to investigate multivalent binding in protein-carbohydrate interactions(Montana State University - Bozeman, College of Letters & Science, 2009) Wolfenden, Mark Leroy; Chairperson, Graduate Committee: Mary J. CloningerPolyvalent interactions in biological systems have been of great interest recently; how nature creates high affinity polyvalent binding with low monomeric affinity, is yet to be clearly understood. We have created a bivalent lectincarbohydrate binding system using dendrimers as the carbohydrate mounted scaffold and Concanavalin A (Con A) as the mannose/glucose binding lectin to investigate this mode of interaction. The relative affinities of the utilized carbohydrates toward Con A are: mannose binds 4 times stronger than glucose, and galactose shows no affinity. With these relative affinities in hand and changing the ratios of mannose, glucose and galactose on the periphery of the PAMAM dendrimer scaffold, we have made a predictable and tuneable system with which to control the polyvalent binding relative affinity. By changing the carbohydrate presentation and varying the size of PAMAM dendrimer used, we can tune the affinity between two orders of magnitude. Although the relative affinities can be predictably altered, the clustering ability across the same generation dendrimer is not affected. In exploring more complex lectin : carbohydrate systems we have made a library of lactose, galactose and galNAc functionalized dendrimers to study binding to galectin-3. This lectin is implicated in numerous cancer related pathway, cellular proliferation and apoptosis. An ELISA based assay was developed to gain binding information of this intruiging interaction. The assay results suggest a reduced effect of binding association even with a large range of monomeric affinities, indicating a multivalent system. The monomer affinities did however affect the lectin recruitment to the dendrimers adsorbed onto a surface. The report here indicated a delicate interplay of modes of multivalent binding that dictate the biological behavior of this important galactose binding lectin.