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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
A new challenge on the horizon is to utilize the large amounts of protein found in the atmosphere to identify different organisms from which the protein originated. Included here is work investigating the presence of identifiable patterns of different proteins collected from the air and biological samples for the purposes of remote identification. Protein patterns were generated using high performance liquid chromatography (HPLC). Patterns created could identify high-traffic and low-traffic indoor spaces. Samples were collected from the air using air pumps to draw air through a filter paper trapping particulates, including large amounts of shed protein matter. In complimentary research aerosolized biological samples were collected from various ecosystems throughout Ecuador to explore the relationship between environmental setting and aerosolized protein concentrations. In order to further enhance protein separation and produce more detailed patterns for the identification of individual organisms of interest; a novel separation device was constructed and characterized. The separation device incorporates a longitudinal gradient as well as insulating dielectrophoretic features within a single channel. This design allows for the production of stronger local field gradients along a global gradient allowing particles to enter, initially transported through the channel by electrophoresis and electroosmosis, and to be isolated according to their characteristic physical properties, including charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. Thus, different types of particles are simultaneously separated at different points along the channel distance given small variations of properties. The device has shown the ability to separate analytes over a large dynamic range of size, from 20 nm to 1 μm, roughly the size of proteins to the size of cells. In the study of different sized sulfate capped polystyrene particles were shown to be selectively captured as well as concentrating particles from 103 to 106 times. Qualitative capture and manipulation of β-amyloid fibrils were also shown. The results demonstrate the selective focusing ability of the technique; and it may form the foundation for a versatile tool for separating complex mixtures. Combined this work shows promise for future identification of individual organisms from aerosolized protein as well as for applications in biomedical research.
ContributorsStaton, Sarah J. R (Author) / Hayes, Mark A. (Committee member) / Anbar, Ariel D (Committee member) / Shock, Everett (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2011
Description
With the advent of the X-ray free-electron laser (XFEL), an opportunity has arisen to break the nexus between radiation dose and spatial resolution in diffractive imaging, by outrunning radiation damage altogether when using single X-ray pulses so brief that they terminate before atomic motion commences. This dissertation concerns the application of XFELs to biomolecular imaging in an effort to overcome the severe challenges associated with radiation damage and macroscopic protein crystal growth. The method of femtosecond protein nanocrystallography (fsPNX) is investigated, and a new method for extracting crystallographic structure factors is demonstrated on simulated data and on the first experimental fsPNX data obtained at an XFEL. Errors are assessed based on standard metrics familiar to the crystallography community. It is shown that resulting structure factors match the quality of those measured conventionally, at least to 9 angstrom resolution. A new method for ab-initio phasing of coherently-illuminated nanocrystals is then demonstrated on simulated data. The method of correlated fluctuation small-angle X-ray scattering (CFSAXS) is also investigated as an alternative route to biomolecular structure determination, without the use of crystals. It is demonstrated that, for a constrained two-dimensional geometry, a projection image of a single particle can be formed, ab-initio and without modeling parameters, from measured diffracted intensity correlations arising from disordered ensembles of identical particles illuminated simultaneously. The method is demonstrated experimentally, based on soft X-ray diffraction from disordered but identical nanoparticles, providing the first experimental proof-of-principle result. Finally, the fundamental limitations of CFSAXS is investigated through both theory and simulations. It is found that the signal-to-noise ratio (SNR) for CFSAXS data is essentially independent of the number of particles exposed in each diffraction pattern. The dependence of SNR on particle size and resolution is considered, and realistic estimates are made (with the inclusion of solvent scatter) of the SNR for protein solution scattering experiments utilizing an XFEL source.
ContributorsKirian, Richard A (Author) / Spence, John C. H. (Committee member) / Doak, R. Bruce (Committee member) / Weierstall, Uwe (Committee member) / Bennett, Peter (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
The properties of materials depend heavily on the spatial distribution and connectivity of their constituent parts. This applies equally to materials such as diamond and glasses as it does to biomolecules that are the product of billions of years of evolution. In science, insight is often gained through simple models with characteristics that are the result of the few features that have purposely been retained. Common to all research within in this thesis is the use of network-based models to describe the properties of materials. This work begins with the description of a technique for decoupling boundary effects from intrinsic properties of nanomaterials that maps the atomic distribution of nanomaterials of diverse shape and size but common atomic geometry onto a universal curve. This is followed by an investigation of correlated density fluctuations in the large length scale limit in amorphous materials through the analysis of large continuous random network models. The difficulty of estimating this limit from finite models is overcome by the development of a technique that uses the variance in the number of atoms in finite subregions to perform the extrapolation to large length scales. The technique is applied to models of amorphous silicon and vitreous silica and compared with results from recent experiments. The latter part this work applies network-based models to biological systems. The first application models force-induced protein unfolding as crack propagation on a constraint network consisting of interactions such as hydrogen bonds that cross-link and stabilize a folded polypeptide chain. Unfolding pathways generated by the model are compared with molecular dynamics simulation and experiment for a diverse set of proteins, demonstrating that the model is able to capture not only native state behavior but also partially unfolded intermediates far from the native state. This study concludes with the extension of the latter model in the development of an efficient algorithm for predicting protein structure through the flexible fitting of atomic models to low-resolution cryo-electron microscopy data. By optimizing the fit to synthetic data through directed sampling and context-dependent constraint removal, predictions are made with accuracies within the expected variability of the native state.
ContributorsDe Graff, Adam (Author) / Thorpe, Michael F. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Matyushov, Dmitry (Committee member) / Ozkan, Sefika B. (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Genomic and proteomic sequences, which are in the form of deoxyribonucleic acid (DNA) and amino acids respectively, play a vital role in the structure, function and diversity of every living cell. As a result, various genomic and proteomic sequence processing methods have been proposed from diverse disciplines, including biology, chemistry, physics, computer science and electrical engineering. In particular, signal processing techniques were applied to the problems of sequence querying and alignment, that compare and classify regions of similarity in the sequences based on their composition. However, although current approaches obtain results that can be attributed to key biological properties, they require pre-processing and lack robustness to sequence repetitions. In addition, these approaches do not provide much support for efficiently querying sub-sequences, a process that is essential for tracking localized database matches. In this work, a query-based alignment method for biological sequences that maps sequences to time-domain waveforms before processing the waveforms for alignment in the time-frequency plane is first proposed. The mapping uses waveforms, such as time-domain Gaussian functions, with unique sequence representations in the time-frequency plane. The proposed alignment method employs a robust querying algorithm that utilizes a time-frequency signal expansion whose basis function is matched to the basic waveform in the mapped sequences. The resulting WAVEQuery approach is demonstrated for both DNA and protein sequences using the matching pursuit decomposition as the signal basis expansion. The alignment localization of WAVEQuery is specifically evaluated over repetitive database segments, and operable in real-time without pre-processing. It is demonstrated that WAVEQuery significantly outperforms the biological sequence alignment method BLAST for queries with repetitive segments for DNA sequences. A generalized version of the WAVEQuery approach with the metaplectic transform is also described for protein sequence structure prediction. For protein alignment, it is often necessary to not only compare the one-dimensional (1-D) primary sequence structure but also the secondary and tertiary three-dimensional (3-D) space structures. This is done after considering the conformations in the 3-D space due to the degrees of freedom of these structures. As a result, a novel directionality based 3-D waveform mapping for the 3-D protein structures is also proposed and it is used to compare protein structures using a matched filter approach. By incorporating a 3-D time axis, a highly-localized Gaussian-windowed chirp waveform is defined, and the amino acid information is mapped to the chirp parameters that are then directly used to obtain directionality in the 3-D space. This mapping is unique in that additional characteristic protein information such as hydrophobicity, that relates the sequence with the structure, can be added as another representation parameter. The additional parameter helps tracking similarities over local segments of the structure, this enabling classification of distantly related proteins which have partial structural similarities. This approach is successfully tested for pairwise alignments over full length structures, alignments over multiple structures to form a phylogenetic trees, and also alignments over local segments. Also, basic classification over protein structural classes using directional descriptors for the protein structure is performed.
ContributorsRavichandran, Lakshminarayan (Author) / Papandreou-Suppappola, Antonia (Thesis advisor) / Spanias, Andreas S (Thesis advisor) / Chakrabarti, Chaitali (Committee member) / Tepedelenlioğlu, Cihan (Committee member) / Lacroix, Zoé (Committee member) / Arizona State University (Publisher)
Created2011
Description
The work described in the thesis involves the synthesis of a molecular triad which is designed to undergo proton coupled electron transfer (PCET) upon irradiation with light. Photoinduced PCET is an important process that many organisms use and the elucidation of its mechanism will allow further understanding of this process and its potential applications. The target compound designed for PCET studies consists of a porphyrin chromophore (also a primary electron donor), covalently linked to a phenol-imidazole (secondary electron donor), and a C60 (primary electron acceptor). The phenol-imidazole moiety of this system is modeled after the TyrZ His-190 residues in the reaction center of Photosystem II (PS II). These residues participate in an intermolecular H-bond between the phenol side chain of TyrZ and the imidazole side chain of His-190. The phenol side chain of TyrZ is the electron transfer mediator between the oxygen evolving complex (OEC) and P680 (primary electron donor) in PSII. During electron transfer from TyrZ to P680*+, the phenolic proton of TyrZ becomes highly acidic (pKa~-2) and the hydrogen is preferentially transferred to the relatively basic imidazole of His-190 through a pre-existing hydrogen bond. This PCET process avoids a charged intermediate, on TyrZ, and results in a neutral phenolic radical (TyrZ*). The current research consists of building a molecular triad, which can mimic the photoinduced PCET process of PSII. The following, documents the synthetic progress in the synthesis of a molecular triad designed to investigate the mechanism of PCET as well as gain further insight on how this process can be applied in artificial photosynthetic devices.
ContributorsPatterson, Dustin (Author) / Moore, Ana L (Thesis advisor) / Gust, Devens (Committee member) / Skibo, Edward B (Committee member) / Arizona State University (Publisher)
Created2011
Description
Alzheimer's Disease (AD) is the sixth leading cause of death in the United States and the most common form of dementia. Its cause remains unknown, but it is known to involve two hallmark pathologies: Amyloid Beta plaques and neurofibrillary tangles (NFTs). Several proteins have been implicated in the formation of neurofibrillary tangles, including Tau and S100B. S100B is a dimeric protein that is typically found bound to Ca(II) or Zn(II). These experiments relate to the involvement of S100B in Alzheimer's Disease-related processes and the results suggest that future research of S100B is warranted. Zn(II)-S100B was found to increase the rate at which tau assembled into paired helical filaments, as well as affect the rate at which tubulin polymerized into microtubules and the morphology of SH-SY5Y neuroblastoma cells after 72 hours of incubation. Zn(II)-S100B also increased the firing rate of hippocampal neurons after 36 hours of incubation. Together, these results suggest several possibilities: Zn(II)-S100B may be a key part of the formation of paired helical filaments (PHFs) that subsequently form NFTs. Zn(II)-S100B may also be competing with tau to bind tubulin, which could lead to an instability of microtubules and subsequent cell death. This finding aligns with the neurodegeneration that is commonly seen in AD and which could be a result of this microtubule instability. Ultimately, these results suggest that S100B is likely involved in several AD-related processes, and if the goal is to find an efficient and effective therapeutic target for AD, the relationship between S100B, particularly Zn(II)-S100B, and tau needs to be further studied.
ContributorsNaegele, Hayley (Author) / Mcgregor, Wade C (Thesis advisor) / Baluch, Debra (Committee member) / Francisco, Wilson (Committee member) / Arizona State University (Publisher)
Created2013
Description
The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (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 goal of this theoretical study of infrared spectra was to ascertain to what degree molecules may be identified from their IR spectra and which spectral regions are best suited for this purpose. The frequencies considered range from the lowest frequency molecular vibrations in the far-IR, terahertz region (below ~3 THz or 100 cm-1) up to the highest frequency vibrations (~120 THz or 4000 cm-1). An emphasis was placed on the IR spectra of chemical and biological threat molecules in the interest of detection and prevention. To calculate IR spectra, the technique of normal mode analysis was applied to organic molecules ranging in size from 8 to 11,352 atoms. The IR intensities of the vibrational modes were calculated in terms of the derivative of the molecular dipole moment with respect to each normal coordinate. Three sets of molecules were studied: the organophosphorus G- and V-type nerve agents and chemically related simulants (15 molecules ranging in size from 11 to 40 atoms); 21 other small molecules ranging in size from 8 to 24 atoms; and 13 proteins ranging in size from 304 to 11,352 atoms. Spectra for the first two sets of molecules were calculated using quantum chemistry software, the last two sets using force fields. The "middle" set used both methods, allowing for comparison between them and with experimental spectra from the NIST/EPA Gas-Phase Infrared Library. The calculated spectra of proteins, for which only force field calculations are practical, reproduced the experimentally observed amide I and II bands, but they were shifted by approximately +40 cm-1 relative to experiment. Considering the entire spectrum of protein vibrations, the most promising frequency range for differentiating between proteins was approximately 600-1300 cm-1 where water has low absorption and the proteins show some differences.
ContributorsMott, Adam J (Author) / Rez, Peter (Thesis advisor) / Ozkan, Banu (Committee member) / Shumway, John (Committee member) / Thorpe, Michael (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2012