Matching Items (342)
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Mutations in the DNA of somatic cells, resulting from inaccuracies in DNA<br/>replication or exposure to harsh conditions (ionizing radiation, carcinogens), may be<br/>loss-of-function mutations, and the compounding of these mutations can lead to cancer.<br/>Such mutations can come in the form of thymine dimers, N-𝛽 glycosyl bond hydrolysis,<br/>oxidation by hydrogen peroxide or other radicals, and deamination of cytosine to uracil.<br/>However, many cells possess the machinery to counteract the deleterious effects of<br/>such mutations. While eukaryotic DNA repair enzymes decrease the incidence of<br/>mutations from 1 mistake per 10^7 nucleotides to 1 mistake per 10^9 nucleotides, these<br/>mutations, however sparse, are problematic. Of particular interest is a mutation in which<br/>uracil is incorporated into DNA, either by spontaneous deamination of cysteine or<br/>misincorporation. Such mutations occur about one in every 107 cytidine residues in 24<br/>hours. DNA uracil glycosylase (UDG) recognizes these mutations and cleaves the<br/>glycosidic bond, creating an abasic site. However, the rate of this form of DNA repair<br/>varies, depending on the nucleotides that surround the uracil. Most enzyme-DNA<br/>interactions depend on the sequence of DNA (which may change the duplex twist),<br/>even if they only bind to the sugar-phosphate backbone. In the mechanism of uracil<br/>excision, UDG flips the uracil out of the DNA double helix, and this step may be<br/>impaired by base pairs that neighbor the uracil. The deformability of certain regions of<br/>DNA may facilitate this step in the mechanism, causing these regions to be less<br/>mutable. In DNA, base stacking, a form of van der Waals forces between the aromatic<br/>nucleic bases, may make these uracil inclusions more difficult to excise. These regions,<br/>stabilized by base stacking interactions, may be less susceptible to repair by<br/>glycosylases such as UDG, and thus, more prone to mutation.
ContributorsUgaz, Bryan T (Author) / Levitus, Marcia (Thesis director) / Van Horn, Wade (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Nucleosomes are the basic repetitive unit of eukaryotic chromatin and are responsible for packing DNA inside the nucleus of the cell. They consist of a complex of eight histone proteins (two copies of four proteins H2A, H2B, H3 and H4) around which 147 base pairs of DNA are wrapped in ~1.67 superhelical turns. Although the nucleosomes are stable protein-DNA complexes, they undergo spontaneous conformational changes that occur in an asynchronous fashion. This conformational dynamics, defined by the "site-exposure" model, involves the DNA unwrapping from the protein core and exposing itself transiently before wrapping back. Physiologically, this allows regulatory proteins to bind to their target DNA sites during cellular processes like replication, DNA repair and transcription. Traditional biochemical assays have stablished the equilibrium constants for the accessibility to various sites along the length of the nucleosomal DNA, from its end to the middle of the dyad axis. Using fluorescence correlation spectroscopy (FCS), we have established the position dependent rewrapping rates for nucleosomes. We have also used Monte Carlo simulation methods to analyze the applicability of FRET fluctuation spectroscopy towards conformational dynamics, specifically motivated by nucleosome dynamics. Another important conformational change that is involved in cellular processes is the disassembly of nucleosome into its constituent particles. The exact pathway adopted by nucleosomes is still not clear. We used dual color fluorescence correlation spectroscopy to study the intermediates during nucleosome disassembly induced by changing ionic strength. Studying the nature of nucleosome conformational change and the kinetics is very important in understanding gene expression. The results from this thesis give a quantitative description to the basic unit of the chromatin.
ContributorsGurunathan, Kaushik (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Woodbury, Neal (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
Description
ABSTRACT The unique structural features of deoxyribonucleic acid (DNA) that are of considerable biological interest also make it a valuable engineering material. Perhaps the most useful property of DNA for molecular engineering is its ability to self-assemble into predictable, double helical secondary structures. These interactions are exploited to design a variety of DNA nanostructures, which can be organized into both discrete and periodic structures. This dissertation focuses on studying the dynamic behavior of DNA nanostructure recognition processes. The thermodynamics and kinetics of nanostructure binding are evaluated, with the intention of improving our ability to understand and control their assembly. Presented here are a series of studies toward this goal. First, multi-helical DNA nanostructures were used to investigate how the valency and arrangement of the connections between DNA nanostructures affect super-structure formation. The study revealed that both the number and the relative position of connections play a significant role in the stability of the final assembly. Next, several DNA nanostructures were designed to gain insight into how small changes to the nanostructure scaffolds, intended to vary their conformational flexibility, would affect their association equilibrium. This approach yielded quantitative information about the roles of enthalpy and entropy in the affinity of polyvalent DNA nanostructure interactions, which exhibit an intriguing compensating effect. Finally, a multi-helical DNA nanostructure was used as a model `chip' for the detection of a single stranded DNA target. The results revealed that the rate constant of hybridization is strongly dominated by a rate-limiting nucleation step.
ContributorsNangreave, Jeanette (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian J.-L. (Committee member) / Seo, Dong Kyun (Committee member) / Arizona State University (Publisher)
Created2011
Description
Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3′ and 5′-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe·BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported.
ContributorsSegerman, Zachary (Author) / Hecht, Sidney M. (Thesis advisor) / Levitus, Marcia (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
This dissertation describes the work on two projects which involves measuring molecular conductance and studying their properties on the nanoscale using various Scanning Tunneling Microscopy (STM) techniques. The first molecule studied was a porphyrin-fullerene moiety known as a molecular Dyad for photovoltaic applications. This project is further divided into two section, the first one involving the characterization of the Dyad monolayers and conductance measurement in the dark. The Dyads are designed to form charge separated states on illumination. The lifetime of the charged states have been measured efficiently but the single-molecule conductance through the molecules have yet to be characterized. The second part of the project describes the set-up of a novel sample stage which enables the study of molecular conductance under illumination. This part also describes the subsequent study of the molecule under illumination and the observation of a unique charge-separated state. It also contains the verification of the presence of this charge-separated using other characterization techniques like transient absorption spectroscopy. The second project described in the dissertation was studying and comparing the predicted rectifying nature of two molecules, identical in every way except for one stereocenter. This project describes the formation of monolayers of the molecule on gold and then studying and analyzing the current-voltage characteristics of the molecules and looking for rectification. Both the molecules proved to be rectifying, one more than the other as predicted by theoretical calculations.
ContributorsBhattacharyya, Shreya (Author) / Lindsay, Stuart (Thesis advisor) / Moore, Ana (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
This thesis focuses on the erotic depictions of Lucretia and Susanna in Renaissance art. Both noted for displaying exemplary chastity, Lucretia and Susanna gained popularity as Christian and secular role models for women in the late Middle Ages and Renaissance. My examination of the heroines addresses the seductive portrayal of these women in painting, which seemingly contradicts the essence of their celebrity. The images specifically analyzed in this thesis include: Lucas Cranach the Elder's Lucretia from 1525, Lucretia from 1533, and Venus from 1532 as well as Tintoretto's Susanna and the Elders and Annibale Carracci's Susanna and the Elders. The scope of my thesis includes both textual and visual analyses of the myths/figures and the disparity that arises between them. Employing Lucretia and Susanna as examples, my aim is to demonstrate a subtle subversion occurring within images of powerful women that ultimately strips them of their power.
ContributorsWilliamson, Jennifer Marie (Author) / Schleif, Corine (Thesis director) / Geschwind, Rachel (Committee member) / Pratt, Rebekah (Committee member) / Barrett, The Honors College (Contributor) / School of Social Transformation (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Art (Contributor)
Created2013-05
Description
Science fiction themed video games, specifically Role Playing Games (RPGs) like Deus Ex: Human Revolution (DX:HR), that focus on an emerging technology, contain features that help to better inform anticipatory governance. In a game like DX:HR, players vicariously experience human-enhancement technology and its societal effects through their in-game character. Acting as the character, the player explores the topic of human-enhancement technology in various ways, including dialogue with non-player characters (NPCs) and decisions that directly affect the game's world. Because Deus Ex: Human Revolution and games similar to it, allow players to explore and think about the technology itself, the stances on it, and its potential societal effects, they facilitate the anticipatory governance process. In this paper I postulate a theory of anticipatory gaming, which asserts that video games inform the anticipatory governance process for an emerging technology. To demonstrate this theory I examine the parts of the anticipatory governance process and demonstrate RPG's ability to inform it, through a case study of Deus Ex: Human Revolution.
ContributorsShedd, Jesse Bernard (Author) / Wetmore, Jameson (Thesis director) / Fisher, Erik (Committee member) / McKnight, John Carter (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2013-05
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional DNA nanostructures. The first difficulty is that the present finite sized template dimension is still very small, usually smaller than 100nm, which will limit the application for large amount of nanomaterials assembly or large sized nanomaterials assembly. Here we tried to solve this problem through developing a new method, superorigami, to construct finite sized DNA structure with much larger dimension, which can be as large as 500nm. The second problem will be explored the ability of DNA structure to assemble inorganic nanomaterials for novel photonic or electronic properties. Here we tried to utilize DNA Origami method to assemble AuNPs with controlled 3D spacial position for possible chiral photonic complex. We also tried to assemble SWNT with discrete length for possible field effect transistor device. In addition, we tried to mimic in vivo compartment with DNA structure to study internalized enzyme behavior. From our results, constructed DNA cage origami can protect encapsulated enzyme from degradation, and internalized enzyme activity can be boosted for up to 10 folds. In summary, DNA structure can serve as an ideal template for construction of functional nanomaterials with lots of possibilities to be explored.
ContributorsZhao, Zhao (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Seo, Dong-Kyun (Committee member) / Arizona State University (Publisher)
Created2013