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Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Molybdenum (Mo) is a key trace nutrient for biological assimilation of nitrogen, either as nitrogen gas (N2) or nitrate (NO3-). Although Mo is the most abundant metal in seawater (105 nM), its concentration is low (<5 nM) in most freshwaters today, and it was scarce in the ocean before 600 million years ago. The use of Mo for nitrogen assimilation can be understood in terms of the changing Mo availability through time; for instance, the higher Mo content of eukaryotic vs. prokaryotic nitrate reductase may have stalled proliferation of eukaryotes in low-Mo Proterozoic oceans. Field and laboratory experiments were performed to study Mo requirements for NO3- assimilation and N2 fixation, respectively. Molybdenum-nitrate addition experiments at Castle Lake, California revealed interannual and depth variability in plankton community response, perhaps resulting from differences in species composition and/or ammonium availability. Furthermore, lake sediments were elevated in Mo compared to soils and bedrock in the watershed. Box modeling suggested that the largest source of Mo to the lake was particulate matter from the watershed. Month-long laboratory experiments with heterocystous cyanobacteria (HC) showed that <1 nM Mo led to low N2 fixation rates, while 10 nM Mo was sufficient for optimal rates. At 1500 nM Mo, freshwater HC hyperaccumulated Mo intercellularly, whereas coastal HC did not. These differences in storage capacity were likely due to the presence in freshwater HC of the small molybdate-binding protein, Mop, and its absence in coastal and marine cyanobacterial species. Expression of the mop gene was regulated by Mo availability in the freshwater HC species Nostoc sp. PCC 7120. Under low Mo (<1 nM) conditions, mop gene expression was up-regulated compared to higher Mo (150 and 3000 nM) treatments, but the subunit composition of the Mop protein changed, suggesting that Mop does not bind Mo in the same manner at <1 nM Mo that it can at higher Mo concentrations. These findings support a role for Mop as a Mo storage protein in HC and suggest that freshwater HC control Mo cellular homeostasis at the post-translational level. Mop's widespread distribution in prokaryotes lends support to the theory that it may be an ancient protein inherited from low-Mo Precambrian oceans.
ContributorsGlass, Jennifer (Author) / Anbar, Ariel D (Thesis advisor) / Shock, Everett L (Committee member) / Jones, Anne K (Committee member) / Hartnett, Hilairy E (Committee member) / Elser, James J (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Introduction: In-store promotion of food products leads to more frequent purchases. Product promotion can vary by store characteristics. We compared marketing strategies used by grocery stores to promote fruit and vegetables (FV) in neighborhoods with varying socio-economic and racial/ethnic characteristics.<br/><br/>Methods: Data was collected from a random sample of 12 large grocery stores from the same national chain located within a 15-mile radius of Downtown Phoenix. Store zip-code level median household income was used to classify stores as located in lower (<$50,000) or higher (>$50,000) income areas. Stores located in neighborhoods with more than 50% Hispanic population were classified as majority Hispanic serving. The ProPromo tool was adapted to document the presence and promotion of FV at 8 distinct locations throughout each store. Types of promotion strategies documented included displays, price promotions, size, or themes.<br/><br/>Results: FV were present at the entrance, islands, checkouts, and produce section; while fruits were promoted in all of these locations, vegetables were promotion in fewer locations. All stores used size and price promotion to promote FV; display was used to promote vegetables in 2 stores and fruits in all stores. On average stores promoted 32 fruits and 38 vegetables. Stores serving higher and lower income areas promoted similar numbers of FV. However, stores in Hispanic majority neighborhoods promoted fewer FV (66) in comparison to those in Hispanic minority areas (73).<br/><br/>Conclusion: Fruit and vegetable promotion disparity associated with neighborhood demographics may contribute to disparities in fruit and vegetable consumption.
ContributorsBrown, Terrell A (Author) / Martinelli, Sarah (Thesis director) / Ohri-Vachaspati, Punam (Thesis director) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Hispanic youth have the highest risk for obesity, making this population a key priority for early childhood interventions to prevent the development of adult obesity and its consequences. Involving parents in these interventions is essential to support positive long-term physical activity and nutrition habits. Interventions in the past have engaged parents by providing information about nutrition and fruit and vegetable intake through written materials or text such as newsletters and text messages. The Sustainability via Active Garden Education (SAGE) intervention used gardening and interactive activities to teach preschool children ages 3-5 about healthy eating and physical activity. It aimed to increase physical activity and fruit and vegetable intake in preschool children as well as improve related parenting practices. The intervention utilized newsletters to engage parents by promoting opportunities to increase physical activity and fruit and vegetable intake for their children at home. The newsletters also encouraged parents to discuss what was learned during the SAGE lessons with their children. The purpose of this paper is to describe the content of the newsletters and determine the parent perception of the newsletters through parent survey responses. This can help inform future childhood obesity interventions and parent engagement.
ContributorsVi, Vinny (Author) / Lee, Rebecca (Thesis director) / Martinelli, Sarah (Committee member) / Edson College of Nursing and Health Innovation (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins.
ContributorsYao, Cheng I Daniel (Author) / Vermaas, Wim (Thesis advisor) / Fromme, Petra (Committee member) / Roberson, Robert (Committee member) / Webber, Andrew (Committee member) / Arizona State University (Publisher)
Created2011
Description
ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure.
ContributorsLawrence, Robert Michael (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian J.L. (Committee member) / Woodbury, Neal W. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Like individual organisms, complex social groups are able to maintain predictable trajectories of growth, from initial colony foundation to mature reproductively capable units. They do so while simultaneously responding flexibly to variation in nutrient availability and intake. Leafcutter ant colonies function as tri-trophic systems, in which the ants harvest vegetation to grow a fungus that, in turn, serves as food for the colony. Fungal growth rates and colony worker production are interdependent, regulated by nutritional and behavioral feedbacks. Fungal growth and quality are directly affected by worker foraging decisions, while worker production is, in turn, dependent on the amount and condition of the fungus. In this dissertation, I first characterized the growth relationship between the workers and the fungus of the desert leafcutter ant Acromyrmex versicolor during early stages of colony development, from colony foundation by groups of queens through the beginnings of exponential growth. I found that this relationship undergoes a period of slow growth and instability when workers first emerge, and then becomes allometrically positive. I then evaluated how mass and element ratios of resources collected by the ants are translated into fungus and worker population growth, and refuse, finding that colony digestive efficiency is comparable to digestive efficiencies of other herbivorous insects and ruminants. To test how colonies behaviorally respond to perturbations of the fungus garden, I quantified activity levels and task performance of workers in colonies with either supplemented or diminished fungus gardens, and found that colonies adjusted activity and task allocation in response to the fungus garden size. Finally, to identify possible forms of nutrient limitation, I measured how colony performance was affected by changes in the relative amounts of carbohydrates, protein, and phosphorus available in the resources used to grow the fungus garden. From this experiment, I concluded that colony growth is primarily carbohydrate-limited.
ContributorsClark, Rebecca, 1981- (Author) / Fewell, Jennifer H (Thesis advisor) / Mueller, Ulrich (Committee member) / Liebig, Juergen (Committee member) / Elser, James (Committee member) / Harrison, Jon (Committee member) / Arizona State University (Publisher)
Created2011
Description
Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed.
ContributorsSpiriti, Justin Matthew (Author) / van der Vaart, Arjan (Thesis advisor) / Chizmeshya, Andrew (Thesis advisor) / Matyushov, Dmitry (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Meteorology is an uncommon term rarely resonating through elementary classrooms. However, it is a concept found in both fourth and sixth grade Arizona science standards. As issues involving the environment are becoming more pertinent, it is important to study and understand atmospheric processes along with fulfilling the standards for each grade level. This thesis project teaches the practical skills of weather map reading and weather forecasting through the creation and execution of an after school lesson with the aide of seven teen assistants.
ContributorsChoulet, Shayna (Author) / Walters, Debra (Thesis director) / Oliver, Jill (Committee member) / Balling, Robert (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12