ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Displaying 1 - 10 of 17
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- All Subjects: Biochemistry
- Creators: Redding, Kevin
Description
There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.
ContributorsReifschneider, Kiera (Author) / Redding, Kevin (Thesis advisor) / Fromme, Petra (Committee member) / Jones, Anne (Committee member) / Arizona State University (Publisher)
Created2013
Description
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is widely accepted as the world's most abundant enzyme and represents the primary entry point for inorganic carbon into the biosphere. Rubisco's slow carboxylation rate of ribulose-1,5-bisphosphate (RuBP) and its susceptibility to inhibition has led some to term it the "bottle neck" of photosynthesis. In order to ensure that Rubisco remains uninhibited, plants require the catalytic chaperone Rubisco activase. Activase is a member of the AAA+ superfamily, ATPases associated with various cellular activities, and uses ATP hydrolysis as the driving force behind a conformational movement that returns activity to inhibited Rubisco active sites. A high resolution activase structure will be an essential tool for examining Rubisco/activase interactions as well as understanding the activase self-association phenomenon. Rubisco activase has long eluded crystallization, likely due to its infamous self-association (polydispersity). Therefore, a limited proteolysis approach was taken to identify soluble activase subdomains as potential crystallization targets. This process involves using proteolytic enzymes to cleave a protein into a few pieces and has previously proven successful in identifying crystallizable protein fragments. Limited proteolysis, utilizing two different proteolytic enzymes (alpha-chymotrypsin and trypsin), identified two tobacco activase products. The fragments that were identified appear to represent most of what is considered to be the AAA+ C-terminal all alpha-domain and some of the AAA+ N-terminal alpha beta alpha-domain. Identified fragments were cloned using the pET151/dTOPO. The project then moved towards cloning and recombinant protein expression in E. coli. NtAbeta(248-383) and NtAbeta(253-354) were successfully cloned, expressed, purified, and characterized through various biophysical techniques. A thermofluor assay of NtAbeta(248-383) revealed a melting temperature of about 30°C, indicating lower thermal stability compared with full-length activase at 43°C. Size exclusion chromatography suggested that NtAbeta(248-383) is monomeric. Circular dichroism was used to identify the secondary structure; a plurality of alpha-helices. NtAbeta(248-383) and NtAbeta(253-354) were subjected to crystallization trials.
ContributorsConrad, Alan (Author) / Wachter, Rebekka (Thesis advisor) / Moore, Thomas (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2012
Description
The heliobacterial reaction center (HbRC) is widely considered the simplest and most primitive photosynthetic reaction center (RC) still in existence. Despite the simplicity of the HbRC, many aspects of the electron transfer mechanism remain unknown or under debate. Improving our understanding of the structure and function of the HbRC is important in determining its role in the evolution of photosynthetic RCs. In this work, the function and properties of the iron-sulfur cluster FX and quinones of the HbRC were investigated, as these are the characteristic terminal electron acceptors used by Type-I and Type-II RCs, respectively. In Chapter 3, I develop a system to directly detect quinone double reduction activity using reverse-phase high pressure liquid chromatography (RP-HPLC), showing that Photosystem I (PSI) can reduce PQ to PQH2. In Chapter 4, I use RP-HPLC to characterize the HbRC, showing a surprisingly small antenna size and confirming the presence of menaquinone (MQ) in the isolated HbRC. The terminal electron acceptor FX was characterized spectroscopically and electrochemically in Chapter 5. I used three new systems to reduce FX in the HbRC, using EPR to confirm a S=3/2 ground-state for the reduced cluster. The midpoint potential of FX determined through thin film voltammetry was -372 mV, showing the cluster is much less reducing than previously expected. In Chapter 7, I show light-driven reduction of menaquinone in heliobacterial membrane samples using only mild chemical reductants. Finally, I discuss the evolutionary implications of these findings in Chapter 7.
ContributorsCowgill, John (Author) / Redding, Kevin (Thesis advisor) / Jones, Anne (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons to be transferred energetically uphill, eventually to be stored as a high energy chemical bond. These protein complexes are highly conserved throughout different photosynthetic lineages and understanding the variations across species is vital for a complete understanding of how photosynthetic organisms can adapt to vastly different environmental conditions. Most knowledge about photosynthesis comes from only a handful of model organisms grown under laboratory conditions. Studying model organisms has facilitated major breakthroughs in understanding photosynthesis, however, due to the vast global diversity of environments where photosynthetic organisms are found, certain aspects of this process may be overlooked or missed by focusing on a select group of organisms optimized for studying in laboratory conditions. This dissertation describes the isolation of a new extremophile cyanobacteria, Cyanobacterium aponinum 0216, from the Arizona Sonoran Desert and its innate ability to grow in light intensities that exceed other model organisms. A structure guided approach was taken to investigate how the structure of photosystem I can influence the spectroscopic properties of chlorophylls, with a particular focus on long wavelength chlorophylls, in an attempt to uncover if photosystem I is responsible for high light tolerance in Cyanobacterium aponinum 0216. To accomplish this, the structure of photosystem I was solved by cryogenic electron microscopy to 2.7-anstrom resolution. By comparing the structure and protein sequences of Cyanobacterium aponinum to other model organisms, specific variations were identified and explored by constructing chimeric PSIs in the model organism Synechocystis sp. PCC 6803 to determine the effects that each specific variation causes. The results of this dissertation describe how the protein structure and composition affect the spectroscopic properties of chlorophyll molecules and the oligomeric structure of photosystem I, possibly providing an evolutionary advantage in the high light conditions observed in the Arizona Sonoran Desert.
ContributorsDobson, Zachary (Author) / Fromme, Petra (Thesis advisor) / Mazor, Yuval (Thesis advisor) / Redding, Kevin (Committee member) / Moore, Gary (Committee member) / Arizona State University (Publisher)
Created2022
Description
All known life requires three main metabolic components to grow: an energy source, an electron source, and a carbon source. For energy, an organism can use light or chemical reactions. For electrons, an organism can use metals or organic molecules. For carbon, an organism can use organic or inorganic carbon. Life has adapted to use any mixture of the endpoints for each of the three metabolic components. Understanding how these components are incorporated in a living bacterium on Earth in modern times is relatively straight forward. This becomes much more complicated when trying to determine what metabolisms may have been used in ancient times on Earth or potential novel metabolisms that exist on other planets. One way to examine these possibilities is by creating genetically modified mutant bacteria that have novel metabolisms or proposed ancient metabolisms to study. This thesis is the beginning of a broader study to understand novel metabolisms using Heliobacteria modesticaldum. H. modesticaldum was grown under different environmental conditions to isolate the impacts of energy, electron, and carbon sources on carbon and nitrogen isotope fractionation. Additionally, the wild type and a novel mutant H. modesticaldum were compared to measure the effects of specific enzymes on carbon and nitrogen isotope fractionation. By forcing the bacterium to adapt to different conditions, variation in carbon and nitrogen content and isotopic signature are detected. Specifically, by forcing the bacterium to fix nitrogen as opposed to nitrogen incorporation, the isotopic signature of the bacterium had a noticeable change. Themutant H. modesticaldum also had a different isotopic signature than the wild type. Without the enzyme citrate synthase, H. modesticaldum had to adapt its carbon metabolic cycle, creating a measurable carbon isotope fractionation. The results described here offer new insight into the effects of metabolism on carbon and nitrogen fractionation of ancient or novel organisms.
ContributorsElms, Nicholas (Author) / Hartnett, Hilairy E (Thesis advisor) / Redding, Kevin (Committee member) / Trembath-Reichert, Elizabeth (Committee member) / Anbar, Ariel D (Committee member) / Arizona State University (Publisher)
Created2021
Description
Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
ContributorsCoe, Jesse (Author) / Fromme, Petra (Thesis advisor) / Sayres, Scott (Thesis advisor) / Mujica, Vladimiro (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
ContributorsRoy Chowdhury, Shatabdi (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
In my thesis, I characterize multi-nuclear manganese cofactors in modified reaction
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
ContributorsEspiritu, Eduardo (Author) / Allen, James P. (Thesis advisor) / Jones, Anne K (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2019
Description
One of the greatest problems facing society today is the development of a
sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts.
To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol.
sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts.
To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol.
ContributorsSommer, Dayn (Author) / Ghirlanda, Giovanna (Thesis advisor) / Redding, Kevin (Committee member) / Moore, Gary (Committee member) / Arizona State University (Publisher)
Created2016
Description
A vast amount of energy emanates from the sun, and at the distance of Earth, approximately 172,500 TW reaches the atmosphere. Of that, 80,600 TW reaches the surface with 15,600 TW falling on land. Photosynthesis converts 156 TW in the form of biomass, which represents all food/fuel for the biosphere with about 20 TW of the total product used by humans. Additionally, our society uses approximately 20 more TW of energy from ancient photosynthetic products i.e. fossil fuels. In order to mitigate climate problems, the carbon dioxide must be removed from the human energy usage by replacement or recycling as an energy carrier. Proposals have been made to process biomass into biofuels; this work demonstrates that current efficiencies of natural photosynthesis are inadequate for this purpose, the effects of fossil fuel replacement with biofuels is ecologically irresponsible, and new technologies are required to operate at sufficient efficiencies to utilize artificial solar-to-fuels systems. Herein a hybrid bioderived self-assembling hydrogen-evolving nanoparticle consisting of photosystem I (PSI) and platinum nanoclusters is demonstrated to operate with an overall efficiency of 6%, which exceeds that of land plants by more than an order of magnitude. The system was limited by the rate of electron donation to photooxidized PSI. Further work investigated the interactions of natural donor acceptor pairs of cytochrome c6 and PSI for the thermophilic cyanobacteria Thermosynechococcus elogantus BP1 and the red alga Galderia sulphuraria. The cyanobacterial system is typified by collisional control while the algal system demonstrates a population of prebound PSI-cytochrome c6 complexes with faster electron transfer rates. Combining the stability of cyanobacterial PSI and kinetics of the algal PSI:cytochrome would result in more efficient solar-to-fuel conversion. A second priority is the replacement of platinum with chemically abundant catalysts. In this work, protein scaffolds are employed using host-guest strategies to increase the stability of proton reduction catalysts and enhance the turnover number without the oxygen sensitivity of hydrogenases. Finally, design of unnatural electron transfer proteins are explored and may introduce a bioorthogonal method of introducing alternative electron transfer pathways in vitro or in vivo in the case of engineered photosynthetic organisms.
ContributorsVaughn, Michael David (Author) / Moore, Thomas (Thesis advisor) / Fromme, Petra (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014