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- All Subjects: Biochemistry
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- 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
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Photosynthesis is a critical process that fixes the carbon utilized in cellular respiration. In higher plants, the immutans gene codes for a protein that is both involved in carotenoid biosynthesis and plastoquinol oxidation (Carol et al 1999, Josse et al 2003). This plastoquinol terminal oxidase (PTOX) is of great interest in understanding electron flow in the plastoquinol pool. In order to characterize this PTOX, polyclonal antibodies were developed. Expression of Synechococcus WH8102 PTOX in E. coli provided a useful means to harvest the protein required for antibody production. Once developed, the antibody was tested for limit of concentration, effectiveness in whole cell lysate, and overall specificity. The antibody raised against PTOX was able to detect as low as 10 pg of PTOX in SDS-PAGE, and could detect PTOX extracted from lysed Synechococcus WH8102. The production of this antibody could determine the localization of the PTOX in Synechococcus.
ContributorsKhan, Mohammad Iqbal (Author) / Moore, Thomas (Thesis director) / Redding, Kevin (Committee member) / Roberson, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Identifying disease biomarkers may aid in the early detection of breast cancer and improve patient outcomes. Recent evidence suggests that tumors are immunogenic and therefore patients may launch an autoantibody response to tumor associated antigens. Single-chain variable fragments of autoantibodies derived from regional lymph node B cells of breast cancer patients were used to discover these tumor associated biomarkers on protein microarrays. Six candidate biomarkers were discovered from 22 heavy chain-only variable region antibody fragments screened. Validation tests are necessary to confirm the tumorgenicity of these antigens. However, the use of single-chain variable autoantibody fragments presents a novel platform for diagnostics and cancer therapeutics.
ContributorsSharman, M. Camila (Author) / Magee, Dewey (Mitch) (Thesis director) / Wallstrom, Garrick (Committee member) / Petritis, Brianne (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor) / Virginia G. Piper Center for Personalized Diagnostics (Contributor) / Biodesign Institute (Contributor)
Created2012-12
Description
Time spent alone is a topic that has been studied in great detail, particularly the manner in which it is spent and the effect it has during the adolescent stage of life. Similarly, stress levels in adolescents have always been a topic of interest because of the effects they could have on the individual later in adulthood. Oddly enough however, the two areas of study have never been looked at in relation to one another. This study will look at different types of alone time as possible stressors in a community sample (N=82) of adolescents transitioning to college. The data on time alone and stress levels was collected through diary reports over a period of 3 days. The analysis only yielded significant effects for females and only for specific categories. It was found that females experience the lowest amount of perceived stress when they are alone and want to be alone, they have more negative affect when their desired environment differs from their current situation, and more positive affect in both the alone incongruence and not alone congruence situations. These results indicate that only women experience stress and affect changes when they encounter different congruent and incongruent environments.
ContributorsVanderwerf, Jennifer (Author) / Doane, Leah (Thesis director) / Knight, George (Committee member) / Arbona, P. Anita (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
Description
This project aims to provide a contextualized history of the Sky Harbor Neighborhood Association‟s community collective action efforts. The Sky Harbor Neighborhood (SHN) of East Phoenix is bounded on the West by 24th St., on the East by 32nd St., on the North by Roosevelt St., and the South by Washington Street. SHN is a majority Latino, low-income, working class community (U.S. Census Bureau, 2010) that faces a variety of challenges including low walkability due to inadequate pedestrian infrastructure, low tree coverage, and crime. East Van Buren St., which has a reputation for being one of Phoenix‟s red-light districts, splits the neighborhood in two. In addition, the SHN lacks some key amenities such as grocery stores and is partly considered a food desert by the United States Department of Agriculture (USDA Economic Research Service, 2012).
ContributorsPearson, Kimberly (Author) / Golub, Aaron (Thesis director) / Wiek, Arnim (Committee member) / York, Abigail (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Sustainability (Contributor)
Created2012-12
Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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