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Item Illuminating dynamic phenomena within organic microstructures with time resolved broadband microscopies(Montana State University - Bozeman, College of Letters & Science, 2024) Hollinbeck, Skyler Robert; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Materials derived from organic chromophore subunits are currently at the forefront of academic and industrial interest. This strong interest is driven in part by the tunability of their extant properties through engineering of both the intra-molecular and inter-molecular structure. The structure of organic materials affects optoelectronic properties because organic chromophores are sensitive to dipole-dipole and charge-transfer coupling interactions. This sensitivity presents both opportunities for tuning functional properties through designing specific packing geometries, and liabilities arising from the disruptive effects of structural disorder. Many organic materials are built from weak noncovalent interactions between chromophores, leading solid-state deposition, and crystallization to be susceptible to microscopic variations in environmental conditions. Structural heterogeneity is regularly intrinsic to organic materials, and even self-assembled systems of covalently linked chromophores exhibit defects. Ergo, in order to disentangle the effects of structural heterogeneity from the inherent properties of the material, the study of organic materials must be anchored with techniques that are capable of correlating optoelectronic properties and excited state evolution with microscale morphological characteristics. We have employed broadband pump-probe microscopies, in conjunction with steady-state and time resolved fluorescence techniques, to examine the effects of structure and coupling on excited state dynamics in solid-state organic microstructures. The study of perylene diimide (PDI) materials revealed that kinetically trapping PDI (KT-PDI) enhanced long-range ordering and led to distinct excited state evolution, delocalized charge-transfer states and long-lived charge separated species. In the MOF PCN-222, excitation energy dependent excited state behavior was observed. Pumping the first excited state (Q-band) led to immobile excited states that were relatively unaffected by local defect densities, whereas pumping the second excited state (Soret-band) led to mobile subdiffusive excited state species whose lifetimes are significantly impacted by morphologically correlated defect sites. Finally, we present progress made toward the construction and utilization of a frequency modulated-femtosecond stimulated Raman microscope, yielding spectra that resolve the location of photoinduced anion formation in KT-PDI. The work presented herein highlights broadband time-resolved microscopy as a potent tool for studying the structure-function relationship and photophysical behavior in molecular solids, deepening our understanding of how structural characteristics influence excited state evolution.Item Changing shape: an investigation into allostery and protein conformational ensembles(Montana State University - Bozeman, College of Letters & Science, 2023) Mattice, Jenna Rose; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Allostery is the mechanism by which action at one site on a protein causes a functional change at a distant site. An allosteric change can manifest as conformational change or a change in protein dynamics. In this way, the study of allostery, protein dynamics, and structural biology are individual, yet related fields. Progress and technical advancements in one field inform and drive the others. In this thesis, four protein complexes 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), acetone carboxylase (AC), Replication protein A (RPA), and Radiation sensitive 52 (Rad52) were studied to elucidate conformational change and allostery during catalysis. A variety of orthogonal biophysical approaches were used to study these systems. To infer changes in protein dynamics and conformation, hydrogen-deuterium exchange coupled to mass spectrometry was used in three of these studies. This technique allows for the probing of the hydrogen bonding network based on ligand binding or mutation. Probing of AC, RPA and Rad52 has led to the description of conformational changes essential for function. Ion mobility coupled to native mass spectrometry was used to investigate the available conformations of 2-KPCC during catalysis and led to the discovery of residues essential for modulating those conformations. The concepts of allostery, conformational ensemble, and protein dynamics have evolved since they were first described. Utilizing mass spectrometry-based techniques, my work helped expand the knowledge of several protein systems that contain allosteric networks which are necessary for function. The studies presented in this thesis increase the understanding not only for these protein systems, but also of protein function on a deeper level.Item 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 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 NMR investigation of non-local effects in a temperature sensitive mutant of the 25 kD tryptophan repressor protein(Montana State University - Bozeman, College of Letters & Science, 2003) Tyler, Robert Charles; Chairperson, Graduate Committee: Valerie Copie