Matching Items (68)
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
As an evolutionary force, hybridization outcomes include introgression, admixture, speciation, and reproductive isolation. While hybridization has been studied in several primates, the marmoset genus Callithrix is an important, but little studied example of Neotropical hybridization. Varying degrees of reproductive isolation exist between Callithrix species, and hybridization occurs at species borders or regions containing introduced and native species. Interbreeding between Callithrix species carries important implications for biodiversity and genetic integrity within the genus. However, species origins and levels of genetic admixture in marmoset hybrid zones are generally unknown, and few population genetic studies of individual Callithrix species exist. Using the mitochondrial control region and 44 microsatellite markers, this work explored the genetic diversity and species origins of two C. penicillata and C. jacchus hybrid zones, as well as genetic diversity and divergence in the parental species. Both marker types showed that C. penicillata is more genetically diverse than C. jacchus. Based on mtDNA, C. jacchus seems to have experienced a past population expansion and C. penicillata evolved under constant population size. The data revealed the existence of a previously undocumented natural hybrid zone along the São Francisco River in NE Brazil and confirmed species origins of an anthropogenic zone in Rio de Janeiro state. The data also showed much lower levels of admixture and genetic diversity within the natural hybrid zone than in the anthropogenic zone. Further, the data suggested that the São Francisco River is an important geographic barrier to gene flow in the natural hybrid zone. On the other hand, admixture patterns within the anthropogenic hybrid zone suggested collapse of reproductive barriers, and the formation of a hybrid marmoset swarm. Thus, this work suggested different evolutionary dynamics in anthropogenic vs. natural animal hybrid zones. Restriction Associated DNA sequencing (RADseq) identified a large number of single nucleotide polymorphisms within C. jacchus and C. penicillata genomes. These preliminary data were used to measure intraspecific genomic diversity and interspecific divergence. In the future, RADseq will be used to study genus-wide diversity of Callithrix species, examine past and present marmoset demographic history, and applied to the evolutionary study of marmoset hybridization.
ContributorsMalukiewicz, Joanna (Author) / Stone, Anne C. (Thesis advisor) / Nash, Leanne (Committee member) / Rosenberg, Michael (Committee member) / Hedrick, Phil (Committee member) / Ruiz-Miranda, Carlo (Committee member) / Arizona State University (Publisher)
Created2013
Description
Malaria is a vector-borne parasitic disease affecting tropical and subtropical regions. Regardless control efforts, malaria incidence is still incredible high with 219 million clinical cases and an estimated 660,000 related deaths (WHO, 2012). In this project, different population genetic approaches were explored to characterize parasite populations. The goal was to create a framework that considered temporal and spatial changes of Plasmodium populations in malaria surveillance. This is critical in a vector borne disease in areas of low transmission where there is not accurate information of when and where a patient was infected. In this study, fragment analysis data and single nucleotide polymorphism (SNPs) from South American samples were used to characterize Plasmodium population structure, patterns of migration and gene flow, and discuss approaches to differentiate reinfection vs. recrudescence cases in clinical trials. A Bayesian approach was also applied to analyze the Plasmodium population history by inferring genealogies using microsatellites data. Specifically, fluctuations in the parasite population and the age of different parasite lineages were evaluated through time in order to relate them with the malaria control plan in force. These studies are important to understand the turnover or persistence of "clones" circulating in a specific area through time and consider them in drug efficacy studies. Moreover, this methodology is useful for assessing changes in malaria transmission and for more efficiently manage resources to deploy control measures in locations that act as parasite "sources" for other regions. Overall, these results stress the importance of monitoring malaria demographic changes when assessing the success of elimination programs in areas of low transmission.
ContributorsChenet, Stella M (Author) / Escalante, Ananias A (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Rosenberg, Michael (Committee member) / Taylor, Jesse E (Committee member) / Arizona State University (Publisher)
Created2014
Description
A phylogenetic revision of the broad-nosed weevil genera Minyomerus Horn, 1876, and Piscatopus Sleeper, 1960 (Entiminae: Tanymecini) is presented. These genera are distributed throughout western North America, from Canada to Mexico and Baja California, primarily in arid and desert habitats, and feed on shrubs such as creosote (Larrea tridentata (DC.) Coville: Zygophyllaceae) and several Asteraceae. Piscatopus was considered monotypic, comprised solely of P. griseus Sleeper, 1960, whereas Minyomerus formerly was comprised of seven species: M. innocuus Horn, 1876 (designated as the type species for Minyomerus in Pierce, 1913), M. caseyi (Sharp, 1891), M. conicollis Green, 1920, M. constrictus (Casey, 1888), M. languidus Horn, 1876, M. laticeps (Casey, 1888), M. microps (Say, 1831). This revision includes comprehensive redescriptions of the previously described species in these genera and descriptions of ten new species: M. imberbus sp. nov., M. caponei sp. nov., M. reburrus sp. nov., M. cracens sp. nov., M. trisetosus sp. nov., M. puticulatus sp. nov., M. bulbifrons sp. nov., M. politus sp. nov., M. gravivultus sp. nov., and M. rutellirostris sp. nov. A cladistic analysis using 46 morphological characters of 22 terminal taxa (5 outgroup, 17 ingroup) was carried out in WinClada and yielded a single most-parsimonious cladogram (length = 82, consistency index = 65, retention index = 82). The monophyly of Minyomerus is supported by the preferred cladogram. The results of the cladistic analysis place Piscatopus griseus within the genus Minyomerus as sister to M. rutellirostris. Therefore, Piscatopus is demoted to a junior synonym of Minyomerus and its sole member P. griseus, is moved to Minyomerus as M. griseus (Sleeper), new combination. Additionally, the species M. innocuus Horn, 1876 is demoted to a junior synonym of M. microps (Say, 1831), based on the principle of priority, and M. microps is elevated to the rank of type for the genus. The species M. languidus, M. microps, and M. trisetosus are putatively considered parthenogenetic, and lack male specimens over a broad range of sampling events. The diversity in exterior and genitalic morphology, range of host plants, overlapping species distributions, and geographic extent suggests an origin during the Miocene (~15 mya).
ContributorsJansen, Michael Andrew (Author) / Franz, Nico M (Thesis advisor) / Wojciechowski, Martin (Committee member) / Rosenberg, Michael (Committee member) / Arizona State University (Publisher)
Created2014
Description
Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
ContributorsKupitz, Christopher (Author) / Fromme, Petra (Thesis advisor) / Spence, John C. (Thesis advisor) / Redding, Kevin (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
Description
The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are required. This dissertation demonstrates a novel complex utilizing earth-abundant Ni in combination with glycine as an efficient catalyst with a modest overpotential of 0.475 ± 0.005 V for a current density of 1 mA/cm2 at pH 11. The production of molecular oxygen at a high potential was verified by measurement of the change in oxygen concentration, yielding a Faradaic efficiency of 60 ± 5%. This Ni species can achieve a current density of 4 mA/cm2 that persists for at least 10 hours. Based upon the observed pH dependence of the current amplitude and oxidation/reduction peaks, the catalysis is an electron-proton coupled process. In addition, to investigate the binding of divalent metals to proteins, four peptides were designed and synthesized with carboxylate and histidine ligands. The binding of the metals was characterized by monitoring the metal-induced changes in circular dichroism spectra. Cyclic voltammetry demonstrated that bound copper underwent a Cu(I)/Cu(II) oxidation/reduction change at a potential of approximately 0.32 V in a quasi-reversible process. The relative binding affinity of Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) to the peptides is correlated with the stability constants of the Irving-Williams series for divalent metal ions. A potential application of these complexes of transition metals with amino acids or peptides is in the development of artificial photosynthetic cells.
ContributorsWang, Dong (Author) / Allen, James P. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
Description
Cyanovirin-N (CVN) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to the N-linked oligosaccharide moiety of the envelope protein gp120. CVN offers a scaffold to develop multivalent carbohydrate-binding proteins with tunable specificities and affinities. I present here biophysical calculations completed on a monomeric-stabilized mutant of cyanovirin-N, P51G-m4-CVN, in which domain A binding activity is abolished by four mutations; with comparisons made to CVNmutDB, in which domain B binding activity is abolished. Using Monte Carlo calculations and docking simulations, mutations in CVNmutDB were considered singularly, and the mutations E41A/G and T57A were found to impact the affinity towards dimannose the greatest. 15N-labeled proteins were titrated with Manα(1-2)Manα, while following chemical shift perturbations in NMR spectra. The mutants, E41A/G and T57A, had a larger Kd than P51G-m4-CVN, matching the trends predicted by the calculations. We also observed that the N42A mutation affects the local fold of the binding pocket, thus removing all binding to dimannose. Characterization of the mutant N53S showed similar binding affinity to P51G-m4-CVN. Using biophysical calculations allows us to study future iterations of models to explore affinities and specificities. In order to further elucidate the role of multivalency, I report here a designed covalent dimer of CVN, Nested cyanovirin-N (Nested CVN), which has four binding sites. Nested CVN was found to have comparable binding affinity to gp120 and antiviral activity to wt CVN. These results demonstrate the ability to create a multivalent, covalent dimer that has comparable results to that of wt CVN.
WW domains are small modules consisting of 32-40 amino acids that recognize proline-rich peptides and are found in many signaling pathways. We use WW domain sequences to explore protein folding by simulations using Zipping and Assembly Method. We identified five crucial contacts that enabled us to predict the folding of WW domain sequences based on those contacts. We then designed a folded WW domain peptide from an unfolded WW domain sequence by introducing native contacts at those critical positions.
WW domains are small modules consisting of 32-40 amino acids that recognize proline-rich peptides and are found in many signaling pathways. We use WW domain sequences to explore protein folding by simulations using Zipping and Assembly Method. We identified five crucial contacts that enabled us to predict the folding of WW domain sequences based on those contacts. We then designed a folded WW domain peptide from an unfolded WW domain sequence by introducing native contacts at those critical positions.
ContributorsWoodrum, Brian William (Author) / Ghirlanda, Giovanna (Thesis advisor) / Redding, Kevin (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2014
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
Description
The AAA+ ATPase Rubisco activase (Rca) regulates the activity of Rubisco, the photosynthetic enzyme responsible for catalyzing biological carbon fixation. However, the detailed mechanism by which Rca self-association controls Rubisco reactivation activity remains poorly understood. In this work, we are using fluorescence correlation spectroscopy (FCS) to better characterize the thermodynamics of the assembly process of cotton Rca. We present FCS data for Rca in the presence of Mg*ATPgS and Mg*ADP and for the D173N Walker B motif mutant in the presence of Mg*ATP. Our data are consistent with promotion and stabilization of hexamers by Mg*ATPgS and Mg*ATP, whereas Mg*ADP facilitates continuous assembly. We find that in the presence of Mg·ADP, Rca self-associates in a step-wise fashion to form oligomeric and higher order forms, with a strong size dependence on subunit concentration. The monomer is the dominant species below 0.5 micromolar, whereas the hexamer appears to be most populated in the 10-30 micromolar range. Large assemblies containing on the order of 24 subunits become dominant above 40 micromolar, with continued assembly at even higher concentrations. Our data are consistent with a highly dynamic exchange of subunits among oligomeric species of diverse sizes. The most likely ADP-mediated assembly mechanism seems to involve the formation of spiral supra-molecular structures that grow along the helical axis by the step-wise addition of dimeric units. To examine the effect of Mg·ATP on oligomerization, we have generated the D173N mutant of Rca, which binds but does not hydrolyze ATP. In range of 8 and 70 micromolar, 60-80% of Rca is predicted to form hexamers in the presence of Mg*ATP compared to just 30-40% with Mg*ADP. We see a clear trend at which hexamerization occurs at high ATP:ADP ratios and in addition, at increasing concentrations of free magnesium ions to 5 milimolar that results in formation of six subunits. We present an assembly model where Mg*ATP promotes and stabilizes hexamerization at low micromolar Rca concentrations relative to Mg*ADP, and suggest that this results from closed ring hexamer formation in Mg*ATP and open hexameric spiral formation in Mg*ADP .
ContributorsKuriata, Agnieszka (Author) / Wachter, Rebekka (Thesis advisor) / Redding, Kevin (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
Description
The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
ContributorsGong, Zhen (Author) / Fromme, Petra (Thesis advisor) / Mor, Tsafrir (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014