Theses and Dissertations at Montana State University (MSU)
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Item Excited-state dynamics of biological molecules in solution: photoinduced charge transfer in oxidatively damaged DNA and deactivation of violacein in viscous solvents(Montana State University - Bozeman, College of Letters & Science, 2017) Beckstead, Ashley Ann; Chairperson, Graduate Committee: Robert WalkerUV radiation from the sun is strongly absorbed by DNA, and the resulting electronic excited states can lead to the formation of mutagenic photoproducts. Decades of research have brought to light the excited-state dynamics of single RNA and DNA nucleobases, but questions remain about the nature of excited states accessed in DNA strands. In this thesis, I present ultrafast spectroscopic observations of photoinduced electron transfer from the oxidatively damaged bases, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine, to adenine in three dinucleotides. The results reveal that charge transfer states are formed on a timescale faster than our instrumental resolution (<0.5 ps), and back electron transfer efficiently returns the excited-state population to the ground state on timescales from tens to hundreds of ps. In addition to recent spectroscopic observations of charge transfer state species in DNA by other groups, our results have augmented understanding of the long-lived transient signals observed in DNA strands. The observation of photoinduced electron transfer in these oxidatively damaged nucleobases also supports a recent proposal regarding the role of oxidative products in pre-RNA catalysis. I discuss these observations in the contexts of fundamental DNA excited-state dynamics and prebiotic chemical evolution. In this thesis, I also present the first ultrafast spectroscopic investigation of violacein, a pigment isolated from Antarctic bacteria. Despite claims for the photoprotective role of this pigment, there has never been a spectroscopic analysis of excited-state deactivation in violacein. Emission spectra, fluorescence quantum yields and excited-state lifetimes of violacein in various solvents were measured for the first time. Both the fluorescence quantum yield and excited-state lifetime of violacein increase in increasingly viscous solvents, suggesting a large-scale motion mediates excited-state deactivation. I compare these results to similar observations of viscosity-dependent excited-state decay rates in other molecules. I also consider the relevance of violacein's excited-state properties to the hypothesized sunscreening role of violacein. Overall, the studies presented in this dissertation illustrate how ultrafast spectroscopic techniques can be used to unravel complex biomolecular excited-state dynamics in solution.Item Ultrafast photochemistry of aqueous iron(III) complexes(Montana State University - Bozeman, College of Letters & Science, 2017) Danforth, Rebecca Ann; Chairperson, Graduate Committee: Erik Grumstrup; Bern Kohler was a co-author of the article, 'Ultrafast photochemical dynamics of hexaaqua iron(III) ion' in the journal 'Chemical physics letters' which is contained within this thesis.The ultrafast photochemical dynamics of aqueous iron(III) solutions were measured utilizing ultrafast pump probe spectroscopy. Aqueous solutions of iron(III) were prepared at low pH (<4.5) and low iron(III) concentration (<5 mM) to allow for small aquairon(III) complexes and ferrihydrite to be studied. Small monomeric and dimeric aquairon(III) complexes were studied to elucidate the mechanisms involved in the formation of OH ° after UV excitation which were previously known to generate OH ° in vastly different quantities. Upon excitation of Fe 3+, a proton is released from a coordinated water molecule to generate FeOH 2+ in less than 200 fs. The newly generated FeOH 2+ can then undergo numerous recombination pathways to regenerate the Fe 3+. Approximately 10% of the excited Fe 3+ undergoes photoreduction and subsequent release of OH ° and Fe 2+ within 20 ps. Exciting FeOH 2+, results in homolysis to form Fe 2+ and OH ° with a wavelength dependent yield with a lifetime of 20 ps. Fe 2(OH) 2 4+ does not appear to generate significant quantities of OH ° however, the dimer is photostable in comparison to Fe 3+ and FeOH 2+. To further the understanding of the primary kinetics of iron(III) in aqueous solutions, ferrihydrite nanoparticles were studied. Ferrihydrite exhibits similar dynamics to hematite in which electrons are excited into the conduction band of ferrihydrite. The electrons can then relax to the bottom of the conduction band within 390 fs before undergoing various recombination process. This limits the amount of iron(III) converted into iron(II) in ferrihydrite. All iron(III) systems studied show unique kinetics after excitation that elucidate the mechanisms behind the generation of OH °.Item Subnanosecond emission from model DNA oligomers characterized through time-correlated single-photon counting spectroscopy(Montana State University - Bozeman, College of Letters & Science, 2017) Skowron, David John; Chairperson, Graduate Committee: Robert Walker; Yuyuan Zhang, Ashley A. Beckstead, Jacob M. Remington, Madison Strawn and Bern Kohler were co-authors of the article, 'Subnanosecond emission dynamics of AT DNA oligonucleotides' in the journal 'Journal of chemical physics and physical chemistry' which is contained within this thesis.Exposure of DNA to UV radiation creates electronic excited states that can decay to mutagenic photoproducts. Excited states can return to the electron ground state through deactivation pathways, preventing photochemical damage. Understanding has significantly advanced over the last decade through the applications of time-resolved techniques capable of picosecond and femtosecond time-resolution. While significant strides have been made towards understanding monomeric deactivation pathways, unraveling the complex photophysics of base multimers still presents a significant challenge. This report uses time-resolved fluorescence and ultrafast transient absorbance to analyze model DNA oligomers to understand how fundamental interactions between monomeric constituents influences the dynamics of base multimers. Model single- and double-stranded DNA oligomers were investigated using the time correlated single photon counting technique to address the uncertainty over how to compare results from time-resolved fluorescent and transient absorption techniques. Emission lifetimes ranging from 50 to 200 ps quantitatively agree with lifetimes measured from transient absorption experiments indicating emission observed on timescales greater than a few picoseconds is the result of excimer or charge recombination luminescence. In attempts to further characterize the time-resolved emission from model oligomers adenine oligomers consisting of 2 and 18 base constituents were examined in aqueous water and heavy water solutions. Differences in dynamics between the two oligomers revealed the average number of bases present within a stacked domain influence the dynamics of these systems. Lifetimes of the emission decays were assigned excimer-like states with various degrees of charge-transfer character. Finally, to further demonstrate the importance of base stacking domain length on the dynamics of these systems, time-resolved emission and absorption of the adenine dinucleotide and 18-mer where examined at temperatures ranging from 7 °C - 80 °C. It was observed that the kinetics between the oligomers was noticeably different at lower temperatures, but not at higher temperatures. It was concluded the domain length of the 18-mer was similar to the domain length of the dinucleotide at high temperatures, but not at low temperatures, demonstrating the domain length significant impacts theS photophysics of DNA.Item Ultraviolet induced decomposition of hexadecanol(Montana State University - Bozeman, College of Letters & Science, 1962) Golden, AnthonyItem Investigating the structural and mechanistic paramters of two radical SAM enzymes, spore photoproduct lyase and HydE(Montana State University - Bozeman, College of Letters & Science, 2013) Ghose, Shourjo; Chairperson, Graduate Committee: Joan B. Broderick; Jonathan K. Hilmer, Kaitlin Duschene, Brian Bothner and Joan B. Broderick were co-authors of the article, 'Solution phase dyanamics of the DNA reoair enzyme spore photoproduct lyase as probed by H/D exchange' submitted to the journal 'FEBS letters' which is contained within this thesis.; Nicholas Boswell, Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Identification and quantitation of a putative substrate for [FE FE]-hydrogenase maturase enzyme HydE' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Mechanistic insights into HydE catalysis' submitted to the journal 'Biochemistry' which is contained within this thesis.The resistance of Bacterial spores to UV radiation makes them a causative agent in many diseases and poses a threat to humans and animals alike. This unique resistance stems from the repair of a thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP)on exposure to UV irradiation. During the early stages of germination, this SP is repaired by an enzyme, spore photoproduct lyase (SPL) into two thymines. SPL is a member of the radical SAM superfamily of enzymes and requires S-adenosylmethionine (SAM) and a [4Fe-4S]1+/2+ cluster to perform its catalysis. The first part of this dissertation is dedicated towards understanding the solution phase dynamics of this protein on binding with its substrate and co-factor via hydrogen deuterium exchange. Analyses of the effects of SAM binding to SPL indicate that the protein does go through a conformational change localized around its active site. We have also demonstrated that the concomitant binding of SAM and dinucleotide SP contributes more significantly to the active site stabilization than what is observed with just SAM binding. Moreover we have provided initial evidence that the SPL might be utilizing the deformation of the phosphodiester back bone of SP to recognize, bind and initiate catalysis. We have unequivocally demonstrated that the catalytic [4Fe-4S] cluster plays a significant role in substrate/cofactor binding most likely due to the stabilization of the 8 residue loop region it resides on. The second part of this dissertation is focused towards understanding the role of maturase proteins in the assembly of the active site of [FeFe]- hydrogenase. The assembly and biosynthesis of the H-cluster requires three accessory enzymes HydE, HydG and HydF. Herein show that HydE utilizes cysteine as a substrate. We have also shown through LCMS and specifically deuterium labeled substrate, that catalysis is initiated via a H atom abstraction from the beta carbon of cysteine. Our investigations into the mechanism of HydG mediated turnover of tyrosine reveal that catalysis is initiated via a single H atom abstraction from the phenolic position of the substrate. Taken together we believe that our investigations have provided some critical insights into specific roles of these enzymes.