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Description
Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for

Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually.

Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.

A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
ContributorsStander, Natasha (Author) / Fromme, Petra (Thesis advisor) / Zatsepin, Nadia (Thesis advisor) / Kirian, Richard (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
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Description
This work aims to characterize protein-nanoparticle interactions through the application of experimental techniques to aid in controlled nanoparticle production for various applications from manufacturing through medical to defense. It includes multiple steps to obtain purified and characterized protein and then the production of nanoparticles using the protein. This application of

This work aims to characterize protein-nanoparticle interactions through the application of experimental techniques to aid in controlled nanoparticle production for various applications from manufacturing through medical to defense. It includes multiple steps to obtain purified and characterized protein and then the production of nanoparticles using the protein. This application of protein requires extremely pure homogenous solution of the protein that was achieved using numerous protein separation techniques which were experimented with. Crystallization conditions, protein separation methods and protein characterization methods were all investigated along with the protein-nanoparticle interaction studies. The main protein of study here is GroEL and the inorganic nanoparticle used is platinum. Some studies on MBP producing gold nanoparticles from an ionic gold precursor were also conducted to get a better perspective on nanoparticle formation. Protein purification methods, crystallization conditions, Car-9 tag testing and protein characterization methods were all investigated along with the focus of this work. It was concluded that more Car9 studies need to be carried out before being used as in the form of a loop in the protein. The nanoparticle experiments were successful and platinum nanoparticles were successfully synthesized using GroEL. The direction of further research in protein-nanoparticle studies are outlined towards the end of the thesis.
ContributorsSirajudeen, Luqmanal Hakim (Author) / Nannenga, Brent L. (Thesis advisor) / Acharya, Abhinav P (Committee member) / Mills, Jeremy H (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Chemical modification of (semi)conducting surfaces with soft-material coatings containing electrocatalysts provides a strategy for developing integrated constructs that capture, convert, and store solar energy as fuels. However, a lack of effective strategies for interfacing electrocatalysts with solid-state materials, and an incomplete understanding of performance limiting factors, inhibit further development. In

Chemical modification of (semi)conducting surfaces with soft-material coatings containing electrocatalysts provides a strategy for developing integrated constructs that capture, convert, and store solar energy as fuels. However, a lack of effective strategies for interfacing electrocatalysts with solid-state materials, and an incomplete understanding of performance limiting factors, inhibit further development. In this work, chemical modification of a nanostructured transparent conductive oxide, and the III-V semiconductor, gallium phosphide, is achieved by applying a thin-film polymer coating containing appropriate functional groups to direct, template, and assemble molecular cobalt catalysts for activating fuel-forming reactions. The heterogeneous-homogeneous conducting assemblies enable comparisons of the structural and electrochemical properties of these materials with their homogeneous electrocatalytic counterparts. For these hybrid constructs, rational design of the local soft-material environment yields a nearly one-volt span in the redox chemistry of the cobalt metal centers. Further, assessment of the interplay between light absorption, charge transfer, and catalytic activity in studies involving molecular-catalyst-modified semiconductors affords models to describe the rates of photoelectrosynthetic fuel production as a function of the steady-state concentration of catalysts present in their activated form. These models provide a conceptual framework for extracting kinetic and thermodynamic benchmarking parameters. Finally, investigation of molecular ‘proton wires’ inspired by the Tyrosine Z-Histidine 190 redox pair in Photosystem II, provides insight into fundamental principles governing proton-coupled electron transfer, a process essential to all fuel-forming reactions relevant to solar fuel generation.
ContributorsWadsworth, Brian Lawrence (Author) / Moore, Gary F (Thesis advisor) / Moore, Thomas A. (Committee member) / Trovitch, Ryan J (Committee member) / Arizona State University (Publisher)
Created2020
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Description
This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified

This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics.

Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.

Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
ContributorsNagaratnam, Nirupa (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
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Description
Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to

Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to be one of the targets against drug design for non-alcoholic fatty liver disease, and more information is needed on the structure of this protein to aid in drug discovery. Here I describe the expression of G0S2 in an E. coli system as well as purification and biophysical characterization of a functional G0S2 in amounts viable for solution state Nuclear Magnetic Resonance (NMR) spectroscopy. Initial spectra of the isotopically labeled protein show well dispersed 15N resonance lines, clean 13C resonances, and dominant a-helices characteristics. These results show that a prepared G0S2 construct is suitable for solution NMR such that 20 amino acids are now assigned in the G0S2 portion of the protein, allowing for further NMR work with this protein for structural discovery. Further work with a large oligomeric complex of G0S2 with Maltose Binding Protein also shows promise for future cryo-EM work.
ContributorsMoran, Michael William (Author) / Fromma, Petra (Thesis advisor) / Guo, Jia (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
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Description
Global industrialization and urbanization have led to increased levels of air pollution. The costs to society have come in the form of environmental damage, healthcare expenses, lost productivity, and premature mortality. Measuring pollutants is an important task for identifying its sources, warning individuals about dangerous exposure levels, and providing epidemiologists

Global industrialization and urbanization have led to increased levels of air pollution. The costs to society have come in the form of environmental damage, healthcare expenses, lost productivity, and premature mortality. Measuring pollutants is an important task for identifying its sources, warning individuals about dangerous exposure levels, and providing epidemiologists with data to link pollutants with diseases. Current methods for monitoring air pollution are inadequate though. They rely on expensive, complex instrumentation at limited fixed monitoring sites that do not capture the true spatial and temporal variation. Furthermore, the fixed outdoor monitoring sites cannot warn individuals about indoor air quality or exposure to chemicals at worksites. Recent advances in manufacturing and computing technology have allowed new classes of low-cost miniature gas sensor to emerge as possible alternatives. For these to be successful however, there must be innovations in the sensors themselves that improve reliability, operation, and their stability and selectivity in real environments. Three novel gas sensor solutions are presented. The first is the development of a wearable personal exposure monitor using all commercially available components, including two metal oxide semiconductor gas sensors. The device monitors known asthma triggers: ozone, total volatile organic compounds, temperature, humidity, and activity level. Primary focus is placed on the ozone sensor, which requires special circuits, heating algorithm, and calibration to remove temperature and humidity interferences. Eight devices are tested in multiple field tests. The second is the creation of a new compact optoelectronic gas sensing platform using colorimetric microdroplets printed on the surface of a complementary-metal-oxide-semiconductor (CMOS) imager. The nonvolatile liquid microdroplets provide a homogeneous, uniform environment that is ideal for colorimetric reactions and lensless optical measurements. To demonstrate one type of possible indicating system gaseous ammonia is detected by complexation with Cu(II). The third project continues work on the CMOS imager optoelectronic platform and develops a more robust sensing system utilizing hydrophobic aerogel particles. Ammonia is detected colorimetrically by its reaction with a molecular dye, with additives and surface treatments enhancing uniformity of the printed films. Future work presented at the end describes a new biological particle sensing system using the CMOS imager.
ContributorsMallires, Kyle Reed (Author) / Tao, Nongjian (Thesis advisor) / Forzani, Erica (Thesis advisor) / Wiktor, Peter (Committee member) / Wang, Di (Committee member) / Alford, Terry (Committee member) / Xian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2020
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Description
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually high number of predicted outer surface lipoproteins of unknown function but with multiple complex roles in pathogenesis, and an unusually low number of predicted outer membrane proteins, given the necessity of bringing in the required nutrients for pathogen survival. Cellular processing of bacterial membrane proteins is complex, and structures of proteins from Bb have all been solved without the N-terminal signal sequence that directs the protein to proper folding and placement in the membrane. This dissertation presents the first membrane-directed expression in E. coli of several Bb proteins involved in the pathogenesis of Lyme disease. For the first time, I present evidence that the predicted lipoprotein, BBA57, forms a large alpha-helical homo-multimeric complex in the OM, is soluble in several detergents, and purifiable. The purified BBA57 complex forms homogeneous, 10 nm-diameter particles, visible by negative stain electron microscopy. Two-dimensional class averages from negative stain images reveal the first low-resolution particle views, comprised of a ring of subunits with a plug on top, possibly forming a porin or channel. These results provide the first evidence to support our theories that some of the predicted lipoproteins in Bb form integral-complexes in the outer membrane, and require proper membrane integration to form functional proteins.
ContributorsRobertson, Karie (Author) / Hansen, Debra T. (Thesis advisor) / Fromme, Petra (Thesis advisor) / Van Horn, Wade (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
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Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
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Description
RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding

RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding of enzymatic pathways. First, sensors for detecting arbitrary target RNAs based the fluorogenic RNA aptamer Broccoli are designed and validated. Studies of three different sensor designs reveal that toehold-initiated Broccoli-based aptasensors provide the lowest signal leakage and highest signal intensity in absence and in presence of the target RNA, respectively. This toehold-initiated design is used for developing aptasensors targeting pathogens. Diagnostic assays for detecting pathogen nucleic acids are implemented by integrating Broccoli-based aptasensors with isothermal amplification methods. When coupling with recombinase polymerase amplification (RPA), aptasensors enable detection of synthetic valley fever DNA down to concentrations of 2 fM. Integration of Broccoli-based aptasensors with nucleic acid sequence-based amplification (NASBA) enables as few as 120 copies/mL of synthetic dengue RNA to be detected in reactions taking less than three hours. Moreover, the aptasensor-NASBA assay successfully detects dengue RNA in clinical samples. Second, RNA scaffolds containing peptide-binding RNA aptamers are employed for programming the synthesis of nonribosomal peptides (NRPs). Using the NRP enterobactin pathway as a model, RNA scaffolds are developed to direct the assembly of the enzymes entE, entB, and entF from E. coli, along with the aryl-carrier protein dhbB from B. subtilis. These scaffolds employ X-shaped RNA motifs from bacteriophage packaging motors, kissing loop interactions from HIV, and peptide-binding RNA aptamers to position peptide-modified NRP enzymes. The resulting RNA scaffolds functionalized with different aptamers are designed and evaluated for in vitro production of enterobactin. The best RNA scaffold provides a 418% increase in enterobactin production compared with the system in absence of the RNA scaffold. Moreover, the chimeric scaffold, with E. coli and B. subtilis enzymes, reaches approximately 56% of the activity of the wild-type enzyme assembly. The studies presented in this dissertation will be helpful for future development of nucleic acid-based assays and for controlling protein interaction for NRPs biosynthesis.
ContributorsTang, Anli (Author) / Green, Alexander (Thesis advisor) / Yan, Hao (Committee member) / Woodbury, Neal (Committee member) / Arizona State University (Publisher)
Created2020
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Description
Proteins are, arguably, the most complicated molecular machines found in nature. From the receptor proteins that decorate the exterior of cell membranes to enzymes that catalyze the slowest of chemical reactions, proteins perform a wide variety of essential biological functions. A reductionist view of proteins as a macromolecular group, however,

Proteins are, arguably, the most complicated molecular machines found in nature. From the receptor proteins that decorate the exterior of cell membranes to enzymes that catalyze the slowest of chemical reactions, proteins perform a wide variety of essential biological functions. A reductionist view of proteins as a macromolecular group, however, may hold that they simply interact with other chemical species. Notably, proteins interact with other proteins, other biological macromolecules, small molecules, and ions. This in turn makes proteins uniquely qualified for use technological use as sensors of said chemical species (biosensors). Several methods have been developed to convert proteins into biosensors. Many of these techniques take advantage of fluorescence spectroscopy because it is a fast, non-invasive, non-destructive and highly sensitive method that also allows for spatiotemporal control. This, however, requires that first a fluorophore be added to a target protein. Several methods for achieving this have been developed from large, genetically encoded autofluorescent protein tags, to labeling with small molecule fluorophores using bioorthogonal chemical handles, to genetically encoded fluorescent non-canonical amino acids (fNCAA). In recent years, the fNCAA, L-(7-hydroxycoumarin-4yl)ethylglycine (7-HCAA) has been used in to develop several types of biosensors.
The dissertation I present here specifically addresses the use of the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) in protein-based biosensors. I demonstrate 7-HCAA’s ability to act as a Förster resonance energy transfer (FRET) acceptor with tryptophan as the FRET donor in a single protein containing multiple tryptophans. I the describe efforts to elucidate—through both spectroscopic and structural characterization—interactions within a 7-HCAA containing protein that governs 7-HCAA fluorescence. Finally, I present a top-down computational design strategy for incorporating 7-HCAA into proteins that takes advantage of previously described interactions. These reports show the applicability of 7-HCAA and the wider class of fNCAAs as a whole for their use of rationally designed biosensors.
ContributorsGleason, Patrick Ray (Author) / Mills, Jeremy H (Thesis advisor) / Hecht, Sidney M. (Committee member) / Fromme, Petra (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2020