Matching Items (25)
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- All Subjects: Biochemistry
- Creators: Redding, Kevin
- Member of: ASU Electronic Theses and Dissertations
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
Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of lower body muscle function. SMA is the second leading genetic cause of death in infants and arises from the loss of the Survival of Motor Neuron (SMN) protein. SMN is produced by two genes, smn1 and smn2, that are identical with the exception of a C to T conversion in exon 7 of the smn2 gene. SMA patients lacking the smn1 gene, rely on smn2 for production of SMN. Due to an alternative splicing event, smn2 primarily encodes a non-functional SMN lacking exon 7 (SMN D7) as well as a low amount of functional full-length SMN (SMN WT). SMN WT is ubiquitously expressed in all cell types, and it remains unclear how low levels of SMN WT in motor neurons lead to motor neuron degradation and SMA. SMN and its associated proteins, Gemin2-8 and Unrip, make up a large dynamic complex that functions to assemble ribonucleoproteins. The aim of this project was to characterize the interactions of the core SMN-Gemin2 complex, and to identify differences between SMN WT and SMN D7. SMN and Gemin2 proteins were expressed, purified and characterized via size exclusion chromatography. A stable N-terminal deleted Gemin2 protein (N45-G2) was characterized. The SMN WT expression system was optimized resulting in a 10-fold increase of protein expression. Lastly, the oligomeric states of SMN and SMN bound to Gemin2 were determined. SMN WT formed a mixture of oligomeric states, while SMN D7 did not. Both SMN WT and D7 bound to Gemin2 with a one-to-one ratio forming a heterodimer and several higher-order oligomeric states. The SMN WT-Gemin2 complex favored high molecular weight oligomers whereas the SMN D7-Gemin2 complex formed low molecular weight oligomers. These results indicate that the SMA mutant protein, SMN D7, was still able to associate with Gemin2, but was not able to form higher-order oligomeric complexes. The observed multiple oligomerization states of SMN and SMN bound to Gemin2 may play a crucial role in regulating one or several functions of the SMN protein. The inability of SMN D7 to form higher-order oligomers may inhibit or alter those functions leading to the SMA disease phenotype.
ContributorsNiday, Tracy (Author) / Allen, James P. (Thesis advisor) / Wachter, Rebekka (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2012
Description
X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease resulting in muscle atrophy and paralysis via degeneration of motor neurons in the spinal cord. In this work, we used X-ray diffraction technique to solve the structures of the three variant of the of SMN protein, namely SMN 1-4, SMN-WT, and SMN-Δ7. The SMN 1-4, SMN-WT, and SMN-Δ7 crystals were diffracted to 2.7 Å, 5.5 Å and 3.0 Å, respectively. The three-dimensional structures of the three SMN proteins have been solved. The FMO protein from Pld. phaeum is a water soluble protein that is embedded in the cytoplasmic membrane and serves as an energy transfer funnel between the chlorosome and the reaction center. The FMO crystal diffracted to 1.99Å resolution and the three-dimensional structure has been solved. In previous studies, double mutant, DX, protein was purified and crystallized in the presence of ATP (Simmons et al., 2010; Smith et al. 2007). DX is a synthetic ATP binding protein which resulting from a random selection of DNA library. In this study, DX protein was purified and crystallized without the presence of ATP to investigate the conformational change in DX structure. The crystals of DX were diffracted to 2.5 Å and the three-dimensional structure of DX has been solved.
ContributorsSeng, Chenda O (Author) / Allen, James P. (Thesis advisor) / Wachter, Rebekka (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2013
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
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 sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar radiation into a readily available and easily transportable form is complex. New materials with optimized physical, electrochemical, and photophysical properties are at the forefront of organic solar energy conversion research. In the work presented herein, porphyrin and organometallic dyes with widely-varied properties were studied for solar energy applications. In one project, porphyrins and porphyrin-fullerene dyads with aniline-like features were polymerized via electrochemical methods into semiconductive thin films. These were shown to have high visible light absorption and stable physical and electrochemical properties. However, experimentation using porphyrin polymer films as both the light absorber and semiconductor in a photoelectrochemical cell showed relatively low efficiency of converting absorbed solar energy into electricity. In separate work, tetra-aryl porphyrin derivatives were examined in conjunction with wide-bandgap semiconductive oxides TiO2 and SnO2. Carboxylic acid-, phosphonic acid-, and silatrane-functionalized porphyrins were obtained or synthesized for attachment to the metal oxide species. Electrochemical, photophysical, photoelectrochemical, and surface stability studies of the porphyrins were performed for comparative purposes. The order of surface linkage stability on TiO2 in alkaline conditions, from most stable to least, was determined to be siloxane > phosphonate > carboxylate. Finally, porphyrin dimers fused via their meso and beta positions were synthesized using a chemical oxidative synthesis with a copper(II) oxidant. The molecules exhibit strong absorption in the visible and near-infrared spectral regions as well as interesting electrochemical properties suggesting possible applications in light harvesting and redox catalysis.
ContributorsBrennan, Bradley J (Author) / Gust, Devens (Thesis advisor) / Moore, Thomas A. (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
Description
Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.
ContributorsWatkins, Jennifer L (Author) / Wachter, Rebekka M. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
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