Dissertations / Theses: 'Structural Insights'

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Gibson, Robert Patrick. "Structural insights into trehalose biosynthesis." Thesis, University of York, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423843.

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Teo, Hsiang Ling. "Structural insights into ESCRT complexes." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614102.

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Busam, Robert Durstberger. "Exonuclease I : structural and biochemical insights/." view abstract or download file of text, 2005. http://wwwlib.umi.com/cr/uoregon/fullcit?p3190508.

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Thesis (Ph. D.)--University of Oregon, 2005.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 83-89). Also available for download via the World Wide Web; free to University of Oregon users.

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McManus, Edward. "Structural insights into lipoate protein ligases." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614075.

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Hunt, James. "Adhesion GPCRs : structural insights into receptor coupling." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590267.

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G-protein coupled receptors (GPCRs) are a diverse superfamily of membrane proteins. They have a wide range of physiological roles and include many successful drug targets. Sequencing of the human genome has revealed a distinct subfamily of GPCRs known as Adhesion GPCRs. These receptors possess unusually large extracellular N-terminal domains which are believed to be involved in cell-cell adhesion. Few data are available which demonstrate that these receptors are able to couple to G-proteins, their classification as GPCRs is primarily based on homology and predicted topology. These receptors are also mainly orphans. This investigation aims to demonstrate G-protein coupling of Adhesion GPCR members and use this coupling to aid de-orphanisation and pharmacological targeting. In this study, a selection of Adhesion GPCRs are expressed in a range of yeast (S. cerevisiae) strains each harbouring different mammalian-yeast chimeric G-proteins. Constitutive coupling of four different Adhesion GPCRs to the chimeric G-proteins is observed via a reporter gene assay. The chimeric G-proteins used represent the human complement, allowing prediction of the G-protein specificities of these receptors. This yeast assay is then used for high throughput screening to identify both potentially native ligands and inhibitors/potential therapeutic compounds. Following analysis in yeast, the Adhesion GPCRs CD97 and EMR2 were expressed in mammalian HEK293 cells where they also displayed constitutive activity when co-expressed with the appropriate Go. subunits (GaJ6).This constitutive activity is strong and mirrors the G-protein specificities seen in yeast. Using this assay, the effects of candidate CD97 and EMR2 ligands were assessed, their putative binding sites

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James, Nathan Rhys. "Structural insights into noncanonical mechanisms of translation." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267783.

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Translation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.

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Cash, Jennifer N. "Structural and Biochemical Insights into Myostatin Regulation." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1313697112.

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Santiago, Cuéllar Julia. "Structural insights into ABA perception and signalling: structure of ABA receptor PYR1." Doctoral thesis, Universitat Politècnica de València, 2011. http://hdl.handle.net/10251/13260.

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La sequía y la salinidad representan estreses ambientales que afectan de forma crítica el crecimiento de las plantas y limitan enormemente su potencial agrícola. La fitohormona ácido abcísico (ABA) juega un papel fundamental en la coordinación de la respuesta y adaptación de las plantas a este tipo de estreses, así como en la regulación del crecimiento y desarrollo vegetal. Elementos intermediarios de la ruta de señalización ya habían sido caracterizados, pero aún se desconocía el mecanismo de percepción y transducción de señal de la hormona. Este trabajo de tesis ha contribuido a la caracterización de una nueva familia de receptores intracelulares de la hormona ABA, formada por 14 miembros y denominada PYR/PYL (de pyrabactin resistance / PYR1-like) /RCAR (de Regulatory Component of Abscisic acid Receptor), y a su caracterización estructural y bioquímica. Estas proteínas son capaces de unir de forma específica la hormona ABA. La unión de la hormona induce en estos receptores un cambio conformacional, que les permite regular la actividad de los reguladores negativos de la ruta: fosfatasas del grupo A como ABI1, ABI2 o HAB1 ( Leung et al., 1994; Meyer et al.,1994; Saez et al., 2004). Para la caracterización de estos receptores se han llevado a cabo abordajes genéticos, bioquímicos, de calorimetría y estudios estructurales.
Santiago Cuéllar, J. (2011). Structural insights into ABA perception and signalling: structure of ABA receptor PYR1 [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/13260
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Lau, Kelvin. "Binding and structural insights of the ryanodine receptor." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50503.

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Ryanodine Receptors (RyR) are large ion channels that are responsible for the release of Ca²⁺ from the sarco/endoplasmic reticulum. The channel consists of a large cytosolic cap which functions as a giant allosteric protein, capable of being modulated by an assortment of binding partners and small molecules. To understand its function and mechanisms one needs to dissect the channel to its smallest parts. Using a combination of isothermal titration calorimetry and x-ray crystallography, two areas have been analyzed: binding by calmodulin (CaM) and the structure of a RyR domain, SPRY2. Calmodulin (CaM) is a Ca²⁺ binding protein that can regulate RyR under conditions of both high and low Ca²⁺ by tuning their Ca²⁺ sensitivity to channel opening and closing in an isoform-specific manner. I analyze the binding of CaM and its individual domains to three different RyR CaM binding regions using isothermal titration calorimetry. I compared binding to skeletal muscle (RyR1) and cardiac (RyR2) isoforms, under both Ca²⁺-loaded and Ca²⁺ free conditions. I find that CaM is able to bind all three regions, but with different binding modes, between the isoforms. Disease mutations target one of the three sites and affect CaM binding and energetics. The SPRY2 domain is one of three repeats of the same fold that are present within the RyR. It has been suggested as a key protein interaction site with dihydropyridine receptors to mediate excitation-contraction coupling in skeletal muscle tissue. RyR1 and RyR2 SPRY2 domains were crystallized and reveal differences with several other known SPRY domain structures. Docking of the RyR1 SPRY2 structure places it in between the central rim and the clamp region. The structure of a disease mutant causing cardiomyopathy is also determined and shows local misfolding. Finally, RyR1 SPRY2 binding to the DHPR II-III loops is undetectable by isothermal titration calorimetry.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

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Velloso, Lucas Malard. "Structural insights into glycoprotein transport and viral escape /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-780-0/.

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Wang, Sheng Verfasser], Oliver [Herausgeber] [Einsle, Roland [Sonstige] Schüle, Manfred [Sammler] Jung, Oliver [Akademischer Betreuer] Einsle, and Roland [Akademischer Betreuer] Schüle. "Structural insights into a novel histone methyltransferase - KMT9." Freiburg : Universität, 2019. http://d-nb.info/1224416538/34.

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Lane-Serff, Harriet. "Structural insights into innate immunity against African trypanosomes." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:3a1415e6-3df4-42dd-827b-d05edb2137be.

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The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A ~50° kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.

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Morgan, Christopher Edward. "Structural and Mechanistic Insights into HIV Regulated Splicing." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1523011774871958.

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Wang, Sheng [Verfasser], Oliver [Herausgeber] Einsle, Roland [Sonstige] Schüle, Manfred [Sammler] Jung, Oliver [Akademischer Betreuer] Einsle, and Roland [Akademischer Betreuer] Schüle. "Structural insights into a novel histone methyltransferase - KMT9." Freiburg : Universität, 2019. http://d-nb.info/1224416538/34.

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Bohm, Raphael. "Structural Insights into Glycan Interactions of Human Pathogens." Thesis, Griffith University, 2014. http://hdl.handle.net/10072/366018.

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Glycans are major components of every cell surface. In addition to their importance in many physiological processes, glycans play a key role during infection of many pathogens. The identification and characterisation of glycan-pathogen interactions at a molecular and atomic level is therefore a crucial step towards the design of novel antimicrobial drugs and vaccines. Protein functions related to glycan interactions include glycan biosynthesis (glycosyltransferases), glycan recognition (lectins) and glycan degradation (glycosylhydrolases). This thesis investigates structure-function relationships of four glycan binding proteins that are important for the infectivity of three major human pathogens: the polysialyltransferase (polyST) of Neisseria meningitidis serogroup B (NmB), the viral protein 8* (VP8*) of rotavirus, and the hemagglutinin (HA) and the neuraminidase (NA) of influenza A virus (IAV).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Martínez, Llinàs Diana. "Structural insights into natural transformation and toxin-antitoxin systems." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/116311.

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La incidència d’infeccions gonocòciques i pneumocòciques roman alta en països en vies de desenvolupament i està augmentant a moltes parts del món. La necessitat de tractaments per a l’individu i per al control de la malaltia a nivel comunitàri és punyent però escollir el tractament adequat ha esdevingut complex com a conseqüència de la capacitat de Neisseria gonorrhoeae i Streptococcus pneumoniae de desenvolupar resistència envers antibiòtics. Aquesta tesi de doctorat investiga mecanismes relacionats amb la transferència genética horitzontal i amb l’arrest del creixement a N. gonorrhoeae and S. pneumoniae. La primera part de la tesi està dedicada a l’estudi de la transformació natural a N. gonorrhoeae i descriu els procediments que han portat a l’expressió, purificació i caracterització bioquímica de ComE, ComA i DprA, tres proteïnes que tenen un paper en la presa i el processament de DNA ambiental. La segona part de la tesi descriu la caracterització estructural mitjançant cristal·lografia de rajos X de RelBE2 de S. pneumoniae, un sistema toxina-antitoxina cromosòmic de tipus II que s’ha relacionat amb la moderació de la traducció i amb l’arrest del creixement en condicions d’escassetat de nutrients, i proposa un model per a la seva regulació.
The incidence of gonococcal and pneumococcal infections remains high in developing countries and is increasing in many parts of the world. The need not only for treatment of the individual but also for control of the diseases at a community level is acute but the selection of appropriate treatments has become a complicated issue by the ability of Neisseria gonorrhoeae and Streptococcus pneumoniae to develop resistance to antibiotics. This PhD thesis investigates mechanisms related to horizontal gene transfer and growth arrest in N. gonorrhoeae and S. pneumoniae. The first part of the thesis is devoted to the study of natural transformation in N. gonorrhoeae and describes the procedures that have lead to the expression, purification and biochemical characterization of ComE, ComA and DprA, three proteins involved in the uptake and processing of environmental DNA. The second part of the thesis describes the structural characterization by X-ray crystallography of S. pneumoniae RelBE2, a chromosomally-encoded type II toxin-antitoxin system that has been linked to translation moderation and growth arrest under starvation conditions, and proposes a model for its regulation.

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Jezyk, Mark R. Sondek John. "Structural insights into the regulation of PLC-[beta]2." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,511.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine. On title page "beta" appears as Greek character.

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Liu, Qian. "Structural insights into apoptotic regulation by BCL-2 family." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95022.

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Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 member, is active in the preservation of mitochondrial integrity during apoptosis. By collective data from nuclear magnetic resonance (NMR) spectroscopy and titration calorimetry, we revealed the selectivity of MCL-1 in binding BH3 ligands of interest to mammalian biology, and proved that the core domain of MCL-1 (cMCL-1) is necessary and sufficient for BH3 ligand binding. We characterized the in vitro protein-protein interaction between cMCL-1 and activated BID, which occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptide. We also present the solution structure of complex cMCL-1:BID-BH3, which may greatly facilitate drug discovery studies of human tumor malignancies. BAK, a multi-region pro-apoptotic protein, directly mediates the mitochondrial outer membrane permeablization (MOMP). We completed a structural investigation of BAK by X-ray crystallography. We report two structures of BAK's homo-dimers, one zinc-mediated (cBAK) and one disulphide-bond-linked (cBAK-o). Their dimerizing sites locate closely at D160 and H164 in cBAK and C166 in cBAK-o, which allow them to compose a unique regulatory element to switch BAK's activity as suggested in mitochondria activity-testing assays. BAK is tightly regulated through protein-protein interactions by MCL-1. We characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment, and studied their interaction in vitro. The non-ionic detergent IGEPAL and the zwitterionic detergent CHAPS have different effects on these two proteins, but both initiate the heterodimerization. The complex of MCL-1 and BAK can be disrupted by either a BID-BH3 peptide, which acts through binding to MCL-1, or a mutation in BH3 region of BAK (L78A), demonstrating the essential role of BAK's BH3 in its regulation by MCL-1. This thesis concludes with a hybrid model for BAK activation:
La protéine MCL-1 (Myeloid cell leukemia 1), qui appartient à la classe de protéines anti-apoptotiques BCL-2, joue un rôle dans le maintien de l'intégrité mitochondriale durant l'apoptose. Les résultats obtenus par résonance magnétique nucléaire (RMN) et par titrage calorimétrique, nous ont permis de mettre en évidence la sélectivité de la protéine MCL-1 pour les ligands mammifères d'interêt biologiques qui contiennent le motif BH3 et nous avons ainsi démontré que le domaine central du facteur MCL-1 (cMCL-1) est nécessaire et suffisant pour cette interaction. Nous avons caractérisé in vitro l'interaction entre le domaine cMCL-1 et le facteur activé BID; cette interaction se produit lentement en solution mais est similaire à celle observée entre le domaine cMCL-1 et le peptide BID-BH3. De plus nous avons résolu la structure du complexe cMCL-1:BID-BH3, qui est une cible potentielle qui pourrait être à la base d'un criblage d'une banque de petites molécules dans le cas de tumeurs humaines malignes. BAK, une protéine pro-apoptotic modulaire, permet la perméabilité de la membrane externe de la mitochondrie: ce mécanisme est dénommé MOMP pour the mitochondrial outer membrane permeablization. Nous avons accompli l'étude structurale de la protéine BAK par cristallographie et diffraction de rayons X. Nous présentons deux complexes de la protéine BAK: un homodimère lié par une molécule de zinc (cBAK) et une qui contient un pont disulfure (cBAK-o). Le site de dimérisation se situe proche des résidu D160 et H164 pour cBAK et C166 pour cBAK-o, ce qui leur confère un élément de régulation unique pour moduler l'activité de BAK comme suggéré dans des essais d'activité mitochondriale. La protéine BAK est finement régulée grâce à son interaction protéine-protéine avec MCL-1. Nous avons caractérisé les changements conformations des facteurs BAK et MCL-1 à l'aide de détergents pour modéliser un environnement m

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Chao, Mao-Hsun. "New insights into structural properties of incommensurate inclusion compounds." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274597.

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Friedmann, David R. "Thermodynamic and structural insights into CSL mediated transcription complexes." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1267132072.

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Byrne, Matthew James. "Structural insights into the biosynthesis of spirotetronate natural products." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687814.

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Spirotetronates are a subclass of polyketide natural products that exhibit an array of potentially exploitable bioactivities. They are characterised by the presence of a tetronic acid ring spiro-linked to cyclohexene. A general route to their biosynthesis has been elucidated through numerous in vitro and in vivo studies. These studies have shown a conserved cassette of enzymes to be responsible for both the incorporation of the eponymous tetronic acid ring, and subsequent chemical modification of this ring. In addition to this, a highly unusual acetylation/deactylation event has been shown to be instrumental in priming spirotetronate intermediates for the [4+2] cyclo-addition that leads to the formation of the spiro-centre. Until recently it remained unclear as to whether or not [4+2] cyclo-addition in the biosynthesis of spirotetronates proceeded via an enzymaticallycatalysed transformation. Herein I will present the crystal structures of the first tetronic acid forming condensing enzyme abyA1, the first acetyl lyase abyA5 and the first reported naturally occurring Diels-Alderase abyU. This work provides a significant insight into the molecular basis of spirotetronate biosynthesis and lays a foundation upon which enzymes from spirotetronate pathways could be used in synthetic biology tbwards the production of non-natural tetronate containing polyketides.

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Dent, Kyle Clayton. "Architecture and assembly of maize streak virus: insights from 3D electron microscopy." Master's thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/13389.

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Includes bibliographical references.
Maize streak virus (MSV), circular single stranded DNA (ssDNA) virus (~2.7kb), is the causative agent of Maize streak disease, and is a devastating pathogen that causes severe crop losses to subsistence farmers in sub-Saharan Africa. MSV is transmitted by the leafhopper Cicadulina mbila, and is the type member of the Mastrevirus genus (family Geminiviridae). MSV shares a unique twinned icosahedral ("geminate") virion architecture (22 x 38 nm) with all other family members. Geminate particles consist of 110 coat protein (CP) subunits that assemble onto a circular single-stranded DNA (ssDNA) genome. Each T= I unit is an incomplete icosahedron assembled from 55 CPs. The structures of MSV and African cassava mosaic virus (ACMV, genus Begomovirus) have been studied by electron cryo- microscopy (cryo-EM) previously. While these investigations revealed some details about the geminate architecture, the interactions of capsid components have not yet been adequately modelled. The two incomplete icosahedral "heads" of the geminate particle are offset from one another and apparently make distinct CP:CP contacts at this region of the virion. Information regarding the nature of quasi- equivalent CP conformers or the sets of amino acid residues that mediate these interactions has not been forthcoming. Since the experimental results of these previous studies are not available in a public database, we were motivated to revisit the structure of MSV in order to obtain a 3D experimental density that might aid pseudo-atomic modelling. The MSV CP:ssDNA interaction has also been shown to be crucial for systemic movement through the host. Hence, quasi-atomic modelling may inform development of antiviral strategies which aim to interfere with virion assembly. MSV virions were isolated from the leaves of maize plants infected by agro-inoculation and visualized in both heavy metal stain and vitreous ice after they had been adsorbed to a thin-layer of continuous carbon to prevent virion aggregation. Virus preparations consisted of distinct CP assemblies consisting of multiples of the incomplete T=I icosahedral unit. Monopartite (icosahedral), bipartite (geminate), tripartite, and higher assemblies were observed suggesting the MSV CP is not only multifunctional but also structurally versatile being able to package ssDNA of variable sizes. Low-dose images were recorded on film, and 3D reconstruction of both monopartite and bipartite capsid species carried out using standard single-particle image processing methodology. The resolution of the bipartite reconstructions was 26 A for the negative-stain dataset, and 23 A for cryo-EM dataset, while the resolution of the monopartite reconstruction was estimated to be ~15 A. Comparative modelling of the MSV CP was undertaken using the pentamer (CPs) of Satellite tobacco necrosis virus (STNV) as a structural template. Correlation-based fitting of icosahedral and geminate atomic models that varied in geometric arrangement of MSV CPs allowed the geometric parameters of the bipartite capsid to be determined. Fitting ofMSV CPs into the EM densities informed our understanding of interfaces which allow the CP to self-associate, and showed that CPs is in fact displaced within the icosahedral geometry of the heads by a 10° rotation about the 5-fold axes of symmetry in comparison to STNV; hence, while quaternary structure of the pentameric capsomer is conserved between these viruses, the quaternary interactions between capsomers of the T=I unit has diverged considerably. This study shows that the offset between the geminate heads of the MSV virion is ~-11°, and that this geometry appears to arise owing to a distinct set of CP:CP interfaces which occur across the equator between two quasi-icosahedral heads and involve regions that would interact to form the CPs: CPs interfaces within each of the heads (across 2-fold and 3-fold symmetry axes). Notably this offset differs from that reported for ACMV, which has a reported offset of 20°. Additionally, the resolution afforded by the icosahedral monopartite reconstruction provided the first structural evidence to suggest that the calcium ion binding site of the STNV CPs (located on the CS axis) is likely to be conserved in MSV. This result suggests that in common with other plant viruses, depletion of calcium ions may be required for genome egress in a newly infected host cell. This study highlights the importance of future high-resolution studies of this unique virion morphology by both X-ray crystallography and cryo-EM.

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Bergen, Konrad [Verfasser]. "Structural insights into DNA polymerases encountering aberrant substrates / Konrad Bergen." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1078230455/34.

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Karathanassis, Dimitrios. "Structural insights into membrane binding by phox homology (PX) domains." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619724.

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Schäfer, Ingmar Bastian. "Structural insights into the function of the Varp/Vamp7 complex." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609037.

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Habenstein, Birgit. "Structural insights into fibrillar proteins from solid-state NMR spectroscopy." Thesis, Lyon 1, 2011. http://www.theses.fr/2011LYO10212.

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La RMN à l’état solide est une méthode de choix pour l’étude des protéines insolubles et des complexes protéiques de haut poids moléculaire. L’insolubilité intrinsèque des protéines fibrillaires, ainsi que leur architecture complexe, rendent difficile leur caractérisation structurale par la cristallographie et par la RMN en solution. La RMN à l‘état solide n’est pas limitée par le poids moléculaire et constitue donc un outil puissant pour l’étude des protéines fibrillaires. L’attribution des résonances RMN est le prérequis pour obtenir des informations structurales à résolution atomique. La première partie de ce travail de thèse décrit le développement de méthodes en RMN à l’état solide pour l’attribution des résonances. Nous avons appliqué ces méthodes afin d’attribuer le domaine C-terminal du prion Ure2 (33 kDa), qui est à ce jour la plus grande protéine attribuée par RMN à l’état solide. Nos résultats fournissent les bases pour l’étude de protéines à haut poids moléculaire à l’échelle atomique. Ceci est démontré dans la seconde partie de ce travail de thèse avec les premières études RMN à l’état solide des fibrilles des prions Ure2 et Sup35. Nous avons caractérisé la structure de ces prions pour les fibrilles entières ainsi que pour les domaines isolés. La troisième fibrille étudiée est l’α- synuclein, fibrille associée à la maladie de Parkinson, pour laquelle nous présentons l’attribution des résonances RMN ainsi que la structure secondaire d’un nouveau polymorphe. Les études présentées ici fournissent de nouvelles clés pour comprendre la diversité des architectures de fibrilles, en considérant les fibrilles comme entités individuelles d’un point de vue structural
Solid-state NMR is the method of choice for studies on insoluble proteins and other high molecular weight protein complexes. The inherent insolubility of fibrillar proteins, as well as their complex architecture, makes the application of x-ray crystallography and solution state NMR difficult. Solid-state NMR is not limited by the molecular weight or by the absence of long-range structural order, and is thus a powerful tool for the 3D structural investigation of fibrillar proteins. The assignment of the NMR resonances is a prerequisite to obtain structural information at atomic level. The first part of this thesis describes the development of solid-state NMR methods to assign the resonances in large proteins. We apply these methods to assign the 33 kDa C-terminal domain of the Ure2p prion which is up to now the largest protein assigned by solid-state NMR. Our results provide the basis to study high molecular weight proteins at atomic level. This is demonstrated in the second part with the first high-resolution solid-state NMR study of Ure2 and Sup35 prion fibrils. We describe the conformation of the functional domains and prion domains in the full-length fibrils and in isolation. The third fibrillar protein addressed in this work is the Parkinson’s disease related α-synuclein whereof we demonstrate the NMR resonance assignment and the secondary structure determination of a new polymorph. Thus, the studies described here provide new insights in the structural diversity of fibril architectures, and plead to view fibrils as individuals from a structural point of view, rather than a homogenous protein family

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Cowper, Ben. "Structural insights into host cell adhesion by Toxoplasma gondii MIC4." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9580.

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Toxoplasma gondii is a highly pervasive protozoan parasite, capable of infecting almost all mammals, including humans. Infection can have fatal consequences for immuno-suppressed individuals, whilst transmission to a developing fetus can induce a spontaneous abortion in pregnant females. T. gondii is perceived to be a model organism in the study of its phylum, Apicomplexa, which includes the Plasmodium species which cause malaria. Apicomplexans are obligate intracellular parasites, in which a strong host cell attachment is established through surface microneme proteins (MICs). Numerous MICs have been identified in T. gondii, many forming multi-adhesive complexes, such as TgMIC1/4/6, within which TgMIC4 is known to possess host cell binding activity within its C-terminal apple-5 & 6 (A56) domains. Prior to these studies, recombinant TgMIC4-A56 was produced yielding a partially-folded protein capable of binding to galactose. Herein the folded component has been identified as A5, and the solution structure of this domain has been solved via NMR spectroscopy. Carbohydrate microarray experiments have confirmed an ability to bind galactosyl-terminated oligosaccharides, whilst NMR and ITC experiments have enabled extensive characterisation of TgMIC4-A5 binding to a range of ligands. The domain binds particularly strongly to a pentasaccharide fragment from GM1 ganglioside; a potential in vivo receptor. Additionally, chemical shift perturbation data and intermolecular NOEs have been used to drive molecular docking of TgMIC4-A5 and lacto-N-biose (Galβ1→3GlcNac). The resulting structure suggests that the mechanism of galactose discrimination by TgMIC4-A5 is similar to that of other galactose-specific lectins. Combined with collaborating studies, this work aids our overall understanding of TgMIC4 function and encourages speculation as to the precise roles of the protein within T. gondii. In addition to a probable contributory role in the initial stages of host cell invasion, the protein may modulate events downstream of this process, through interactions with galactosylated receptors.

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Polano, Maurizio. "Structural insights into alternate aggregation states of mouse prion protein." Doctoral thesis, SISSA, 2009. http://hdl.handle.net/20.500.11767/4765.

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Gucinski, Ashley Christine. "Gas Phase Structural Studies of Peptide Fragment Ions: Structural Insights into Mass Spectrometry Fragmentation Mechanisms." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202766.

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This dissertation presents extensive structural studies of gas-phase peptide fragment ions, with a specific focus on b₂⁺ ions. Fragment ion structures can provide important insights into peptide fragmentation mechanisms. Based on the structures formed, information about the preference of competing b ion formation pathways can be obtained. b₂⁺ ion structures are of interest because of their large relative abundances in MS/MS spectra, which are difficult to predict. Prior to this work, only a few b₂⁺ ion structures were determined; these systems featured only aliphatic residues and all formed oxazolones. The work presented herein examines the influence of basic, acidic, and backbone-attached sidechains on peptide fragmentation mechanisms, as revealed by the resulting b₂⁺ fragment ion structure(s) formed. Specifically, the structures of several histidine, aspartic acid, and proline-containing b₂⁺ ions are determined by using action IRMPD spectroscopy, fragment ion HDX, and DFT calculations. The structures of a series of histidine analogue-containing b₂⁺ ions reveal that the location and availability of the pi-nitrogen is essential for diketopiperazine formation. The histidine sidechain bulk or strain interferes with the complete trans-cis isomerization required for diketopiperazine formation, so the oxazolone structure is also present. Xxx- Pro b₂⁺ ions favor oxazolone formation with aliphatic N-terminal residues. HP favors the diketopiperazine, combining the histidine effect and the proline cis conformation propensity. For Xxx-Asp b₂⁺ ions, aspartic acid significantly influences b₂⁺ ion structure only with an N-terminal histidine or lysine; both HD and KD form a mixture of oxazolone, anhydride, and diketopiperazine structures, presenting the first spectroscopic evidence for the anhydride b₂⁺ion structure. The HA and AH b₂⁺ ions feature the same structures, but HP and PH do not, showing that residue position matters. Additionally, while relative intensities and HDX rates featured some fluctuation, peptide precursor composition differences did not alter the mixture of b₂⁺ ion structures formed for a given b₂⁺ ion. To complement existing gas-phase structural methods, the utility of a new technique, QCID-HDX-IRMPD, was applied to m/z 552.28 from YAGFL-OH. Both the standard b₅⁺ fragment ion and an isobaric non-C-terminal water loss ion are present. Without separation of these isomers, MS/MS spectral interpretation would be complicated.

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Spivak, Mariano Alejo. "Electronic structure calculations on extended metal atom chains. Insights on structural, magnetic and transport properties." Doctoral thesis, Universitat Rovira i Virgili, 2017. http://hdl.handle.net/10803/399580.

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En aquest treball, es van utilitzar diferents mètodes computacionals per estudiar les propietats de cadenes esteses de metalls de transició (EMACs en anglès). Es va simular la flexibilitat estructural de cadenes de tres àtoms de crom, amb CASSCF/CASPT2 i es van identificar estructures simètriques i asimètriques en un entorn de baixa energia. Basats en aquests resultats, vam realitzar dinàmiques moleculars de primers principis (AIMD) per entendre l'efecte de l'energia tèrmica i com aquesta modifica la proporció d'estructures. També es van estudiar els enllaços metall-metall en compostos de crom, utilitzant el model d'ordre d'enllaç efectiu (EBO) amb els números d'ocupació naturals de la funció d'ona CASSCF. Es van calcular constants d'acoblament magnètic per a compostos bimetàl·lics i EMACs de níquel mitjançant dues estratègies. MC-PT2 amb espai actiu mínim utilitzant orbitals moleculars millorats a partir d'un càlcul d'estats-mitjanats, i es va utilitzar un mètode nou (MCPDFT) per al magnetisme de EMACs grans, que ha mostrat bons resultats en el compost de cinc níquels. Finalment, estudiem propietats del transport d'electrons per dos EMACs de ruteni. Proposem l'ús d'un elèctrode gate metàl·lic per modular els nivells moleculars dels compostos i obtenir espècies redox actives. També utilitzem un mètode químicament més intuïtiu, que proposa crear parells iònics dins de la cel·la.
En este trabajo, se utilizaron diferentes métodos computacionales para estudiar las propiedades de cadenas extendidas de metales de transición (EMACs en inglés). Se simuló la flexibilidad estructural de cadenas de tres átomos de cromo, con CASSCF/CASPT2 y se identificaron estructuras simétricas y asimétricas en un entorno de baja energía. Basados en estos resultados, realizamos dinámicas moleculares de primeros principios (AIMD) para entender el efecto de la energía térmica y como ésta modifica la proporción de estructuras. También se estudiaron los enlaces metal-metal en compuestos de cromo, utilizando el modelo de orden de enlace efectivo (EBO) con los números de ocupación naturales de la función de onda CASSCF. Se calcularon constantes de acoplamiento magnético para compuestos bimetálicos y EMACs de níquel mediante dos estrategias. MC-PT2 con espacio activo mínimo utilizando orbitales moleculares mejorados a partir de un cálculo de estados-promediados, y se utilizó un método nuevo (MCPDFT) para el magnetismo de EMACs grandes, que ha mostrado buenos resultados en el compuesto de cinco níqueles. Finalmente, estudiamos propiedades del transporte de electrones para dos EMACs de rutenio. Proponemos el uso de un electrodo gate metálico para modular los niveles moleculares de los compuestos y obtener especies redox activas. También utilizamos un método químicamente más intuitivo, que propone crear pares iónicos dentro de la celda.
In this work we use different computational methods in the study of the properties of Extended Metal Atom Chains. The structural flexibility of trichromium chains has been simulated with CASSCF/CASPT2 and symmetric and asymmetric structures were identified in an extremely flat energy landscape. Based on these results, Ab initio molecular dynamic simulations were performed to understand how the thermal energy modifies the proportion of cited structures. In addition, the metal-metal bonding of chromium compounds was characterized using the Effective Bond Order (EBO) model with the natural occupation numbers of the CASSCF wave function. Furthermore, magnetic coupling constants were computed for nickel bimetallic and EMACs compounds, using two different approaches. Minimal active space MC-PT2 was performed with improved molecular orbitals based on state-average calculations, and a recently developed method (MCPDFT) used for the magnetism of large EMACs, showing good results in the five-nickel compound. Finally, the electron transport properties were simulated for two ruthenium EMACs. We propose the use of a metallic gate electrode to modulate the molecular levels of the compounds and achieve redox active species. In addition, another more chemically intuitive approach was tested, that consist of forming an ionic pair in-situ.

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Mitchell, Carter Alexander. "Structural, functional, and computational insights into the ANL superfamily of enzymes." Thesis, State University of New York at Buffalo, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3598714.

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Members of the ANL superfamily of enzymes are involved in primary and secondary metabolism throughout all domains of life and identify key pathways that contribute to essential physiological reactions as well as defense mechanisms to evade competition. Specifically, acetyl-CoA synthetases are directly involved in energy metabolism, while NonRibosoaml Peptide Synthetases and some Aryl-CoA Ligases produce secondary natural products that confer virulence for the producing organism. Due to the ANL superfamily's ubiquitous involvement in primary and secondary metabolism, gaining an understanding of how these enzymes work and identifying ways to regulate them could provide an alternative route for antibiotic targets. It is well documented that domain alternation is paramount for the ANL superfamily of enzymes including the adenylation and thioester-forming reactions of NRPS adenylation domains. This thesis utilizes structural and functional analysis in conjunction with computational methods to further our understanding of these unique enzymes.

In chapter 2 we present the structure of an adenylation:Peptidyl Carrier Protein di-omain NRPS from the cryptic PA1221 biosynthetic operon from Pseudomonas aeruginosa. The PA1221 structure is the second example of an adenylation:PCP in the PDB and validates the chimeric fusion interactions of EntE-B. The similar interacting regions are between the 2^nd PCP helix and a helix in the N-terminal subdomain of the adenylation domain as well as the loop connecting the longest β-strands of the C-terminal subdomains interacting with loop 1 of the PCP.

Chapter 3 presents the structure of an acetoacetatyl-CoA Synthetase that is a confirmed substrate for a protein acetyltransferase, PatA, for inactivation through acetylation of the catalytic A10 lysine. This Streptomyces lividans acetoacetyl-CoA synthetase is the first structure to fully resolve the loop connecting C-terminal extension helix to the C-terminal subdomain. The C-terminal extension is only present in ACS proteins revealing an interaction where the C-terminal extension stabilizes the dynamic P-loop in the adenylate forming conformation.

In chapter 4 we further explore the PA1221 operon by functionally identifying the substrate preference of PA1215, the hypothetical fatty-acyl-CoA Ligase, that is proposed to acylate the charge PCP of PA1221. We computationally validate the substrate preference with a homology model and AutoDock to gain insight into the proteins slow kinetics. We also provide further insight into the biochemistry of a subset of ANL superfamily members, the phenylacetic acid CoA ligases, involved in the utilization of aryl-carboxylic acids as a carbon source as well as the derivatization of penicillin. We analyze their unique dimeric structures identifying structural motifs that are contributed through the dimeric interface, but are otherwise located to different sides of the enzyme in a monomeric form.

Finally, to help identify how the protein moves between the two productive conformations we subject members of the superfamily to computational dynamic simulations including Anisotropic Network Modeling, Interpolative Elastic Network Modeling, all-atom molecular dynamics, and analyze the output from these methods with Principal Component and Normal Mode Analysis. We developed a method to visualize a dynamic reaction coordinate through measuring the Conformation Determining Angle (defined by structural motifs that are present in superfamily members) and use this metric to interrogate all ANL superfamily member PDB entries for domain organization. Finally, we test our hypothesis that domain alternation proceeds through an extended, open conformation with structural comparisons and MD. Here we report functional and structural analysis of ANL superfamily members that are related through bacterial cell metabolism and natural product biosynthesis.

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32

Charlton, Ian David. "New insights into micellar structural evolution and interaction using voltammetric methods." Thesis, University of Newcastle Upon Tyne, 1999. http://hdl.handle.net/10443/798.

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The development of electrochemical techniques as applied to self-assembled supramolecular systems (e. g. micelles) has advanced over the past decade. The main properties that have been elucidated by these techniques have been micellar selfdiffusion and size. Although there are reports that have paid attention to the qualitative influence of intermicellar interactions on the behaviour of the micellar system, quantitative assessments of interaction are very limited. In this thesis, the application of rotating disk voltammetry, primarily, has led to a quantitative rationalisation of intermicellar interactions in cationic and nonionic micellar systems over a range of surfactant and electrolyte concentrations. Two `normal' micellar systems are studied with aggregates formed from cationic (CTAC) and nonionic (Triton X-100) surfactants. Initial measurements and analysis yields micellar sizes that are consistent with published values, demonstrating the validity and the ease of application of electrochemical techniques. Measuring self-diffusion coefficients over a range of electrolytes provides a comprehensive assessment of micellar phase behaviour and yields further structural parameters which are conventionally determined using a variety of methods. The first reported study of electrochemistry in a reverse micelle `nanoemulsion' is presented. The growth in micellar size on the addition of a solubilised probe gives important inferences for the careful control of particle growth in a reverse micelle `nano-reactor'. In summary, the thesis, as the title states, gives new insights pertaining to micellar structural evolution and interaction. The thesis will examine the benefits of applying electrochemical techniques to study micellar systems and concentrate, predominately, on the wealth of information that can be obtained by the resultant analysis. The work forms an excellent basis for not only further quantitative analysis but also as a phenomenological template for employment in the study of a diversity of self-assembled supramolecular species.

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33

MacLellan, Jonathan G. "Structural insights into superbase chemistry and related inverse crown base systems." Thesis, University of Strathclyde, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275156.

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Flatt, Justin Wayne. "STRUCTURAL INSIGHTS INTO RECOGNITION OF ADENOVIRUS BY IMMUNOLOGIC AND SERUM FACTORS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1387451692.

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35

Sui, Xuewu. "Structural and biochemical insights into catalytic mechanisms of carotenoid cleavage oxygenases." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1473258604663537.

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36

Leen, Eoin. "Structural insights into the transcription and translation of murine norovirus RNA." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11173.

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Noroviruses are positive-sense single-stranded RNA viruses which, in humans, cause rapid onset diarrhoea and vomiting. There is an estimated 21 million cases of noroviral gastroenteritis in the United States per year. The work in this thesis has focused on two noroviral proteins, the viral protein genome-linked (VPg), and the viral RNA dependent RNA polymerase (NS7pol). The VPg protein is covalently linked to the 5' end of the noroviral genome and is a key component of translation and replication initiation in noroviruses. Using Nuclear magnetic resonance spectroscopy (NMR) we have analysed the murine norovirus (MNV) and a human norovirus VPg (Lordsdale virus (LDV)) proteins. The VPg protein of both viruses has a small structured helical core with extensive N and C-terminal flexible regions. We has also determined the structure of the MNV NS7pol as well as two high fidelity mutants (P72S and E75S) using X-ray crystallography. The NS7pol protein has a very similar “right drinking hand” structure to other RNA dependent RNA polymerases. The fidelity mutants were structurally identical to the wild type protein but had subtle changes in local hydrogen bonding networks. This is consistent with similar studies performed with picornaviral polymerases. In addition we used NMR and surface plasmon resonance to characterise the interaction of the MNV VPg protein with the NS7pol protein. The interaction between full length VPg and MNV NS7pol is weak, (KD ~160 μM). In addition the NS7pol interacts with both the full length VPg and the core domain of this protein.

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Lougher, Matthew J. "Functional and structural insights into MmyJ, An ArsR-like transcriptional repressor." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/77520/.

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MmyJ is a protein encoded in the methylenomycin antibiotic gene cluster in Streptomyces coelicolor A3(2). It was identified as a novel member of the ArsR family of transcription repressors. In depth bioinformatic analyses were carried out to compare this to other members of the ArsR family, leading to high confidence of its classification. An expression system was engineered to investigate MmyJ in vitro, leading to the discovery of the presence of a covalently bonded dimer, which is unusual for ArsR family proteins and as such was thought to be an artefact of purification. A C49S mutant was then also engineered without the capacity to form covalent dimers, such that the two variants could be investigated side by side. Work was carried out to investigate the stability of MmyJ, and it was found that its secondary structure denatured above temperatures of 40oC and that the protein is only robust to a single freeze/thaw cycle. Electrophoretic mobility shift assays were then used to prove that MmyJ binds specifically to a 13-1-13 semi conserved inverted repeat overlapping the -35 region of both mmyJ and mmr promoters. This indicates that MmyJ not only regulates its own expression, but also that of Mmr, an efflux pump that removes methylenomycin A from the cell upon biosynthesis. It was also demonstrated that methylenomycin A caused complete dissociation of the MmyJ:DNA complex when present in a 20 times molar excess, hence suggesting that the methylenomycin A resistance mechanism is triggered in the presence of methylenomycin A. This is the first reported instance of an ArsR family protein sensing a non-metallic ligand. Structural insights into MmyJ were also sought, with a homology model produced by Phyre2 analysis complimented by circular dichroism analysis of recombinant MmyJ. X-ray diffraction data were obtained to a resolution of 2.1 A over 300o, but the phase of these data has yet to be determined, so the crystal structure has not yet been solved.

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Varzandeh, Simon. "Structural and biochemical insights into the ATP-dependent chromatin remodeler LSH." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29515.

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Chromatin remodelling proteins support a variety of cellular functions and utilise the energy from ATP hydrolysis to either reposition or evict nucleosomes. One such protein, Lymphoid specific helicase (LSH), regulates DNA methylation in mammalian cells cooperatively with DNA Methyltransferase 3B (DNMT3B) through binding of the N-terminal domain of LSH. The correct functioning of LSH is essential for heterochromatin formation, with a knockout of LSH causing perinatal lethality or severe developmental abnormalities. There is little biochemical data and no structural data on LSH. Therefore, we aim to determine the structural characteristics and regulatory mechanism of LSH in vitro. LSH was expressed in an optimised insect cell system which increased protein yield 25-fold with greater than 95% purity. LSH is monomeric with increased thermal stability upon ATP or ADP binding. Full length LSH could not be crystallised therefore a core ATPase region of LSH missing the N-terminal domain was identified through limited proteolysis. This also provided evidence the N-terminal domain of LSH is disordered, which was proven through biophysical characterisation of LSH1-176. Expression of the LSH ATPase region was weak and the protein was unstable; suggesting the N-terminal domain of LSH is required for LSH stability. Therefore, complementary structural methods were used to study LSH. Crosslinking mass-spectrometry revealed the N and C termini are in close proximity, suggesting flexible linking regions, which was supported by limited proteolysis experiments. Negative staining Electron Microscopy defined LSH as a tri-lobal and elongated structure which could harbour the ATPase region in the two spherical lobes. 3D modelling of SAXS data obtained of LSH was in agreement with EM data. To understand molecular mechanisms of LSH, functional studies investigating LSH:DNA and LSH:DNMT3B interactions were performed. LSH had a KD for dsDNA of 0.4 μM in solution. LSH does not bind ssDNA nor does it have a greater affinity for methylated dsDNA. LSH was found to bind the dsDNA overhangs of nucleosomes but not to core nucleosomes, suggesting LSH solely interacts with DNA in chromatin and not histones. A stable complex of LSH:DNMT3B could not be achieved in vitro, however, other components for complex formation may have been missing. This study has improved our understanding of LSH structure, biophysical properties and its biochemical interaction with DNA and nucleosomes. This study has laid the foundations for the structural investigations of a LSH:nucleosome and potentially a LSH:DNMT3B complex in vitro to gain a greater understanding of how functional domains of LSH regulates its enzymatic function.

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Giorgetti, Alejandro. "Structural predictions of HCN/CNG ion channels: Insights on channels' gating." Doctoral thesis, SISSA, 2004. http://hdl.handle.net/20.500.11767/4655.

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Evolution built a membrane around the earliest forms of life in order to isolate them from the external environment. The cell membrane is constituted by two layers of phospholipids, which are molecules having a polar head and non-polar tails. Two films of these molecules are assembled together by hydrophobic forces building a very stable lipid bilayer. Inserted in this amphiphilic environments are membrane proteins, which have both hydrophobic and hydrophilic regions on their surface. In highly evolved and specified cells this class of proteins carries out a variety of different activities essential for the cell and organism life, like the antibody recognition in lymphocytes and the nervous pulse transmission in neurons. Although the presence of the membrane helps cells to retain vital ingredients, it prevents the access to necessary ionized substrates and ions, because the hydrophobic core is a high free energy barrier in the diffusion of charged molecules. Membrane spanning pores are a common feature to ionic channels (Hille, 2001;Chang et al., 1998), and they are presents in different classes of other biological transporter proteins like bacterial porins and aquaporins. Special membrane proteins, the ionic channels, form holes through the cell membrane, providing a feasible path for ion exchanges.

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Paoletti, Francesca. "Structural and functional insights into the biological function of mouse proNGF." Doctoral thesis, SISSA, 2006. http://hdl.handle.net/20.500.11767/4829.

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In the recent years one of the lines of in vestigation in the Neur obiology Laboratory at SISSA has been to study the molecular det erminants of Alzheimer’s Disease (AD), one of the most known and diffused neur odegenerative aging diseases. AD is characterized by lesions in the brai n cortex, including the presence of β -amyloid plaques and neurofibrillary tangles c ontaining phosphorylated tau protein. In Alzheimer’s disease neuronal degeneration is found in selected areas of the brain, in particular in cortical, hippocam pal and basal forebrain cholinergic neurons (reviewed in Price et al ., Ann. Rev. Neurosci., 1986) . The investigation on the involvement of the neurotrophin Nerve Gr owth Factor (NGF) in AD has been extensive, because it promotes the survival and regulates the function of cholinergic neurons of the basal forebr ain. (reviewed in Counts et al. , J. Neuropath. Exp. Neuro., 2005). NGF is translated as a pre- pro-protein, proNGF, the im portance of which in the recent years has grown much, thanks to impor tant findings on its bi ological functions, besides the one of promoting protein foldin g. Accordingly, the increasing number of involved new actors has complicated also t he scenario of the investigations on the molecular determinants in AD.

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Li, Lichun. "Insights into subgenomic RNA synthesis in coronaviruses from structural and biophysical studies." [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-2431.

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Jackson, Pilgrim J. "Structural insights into appetite : investigations of the ligands involved in melanocortin signaling /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2005. http://uclibs.org/PID/11984.

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43

Varadan, Ranjani. "NMR studies of polyubiquitin chains insights into structural basis of functional diversity /." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1959.

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Thesis (Ph. D.) -- University of Maryland, College Park, 2004.
Thesis research directed by: Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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Johnston, Christopher Alan Siderovski David P. "Structural insights into the molecular basis of heterotrimeric G-protein signal transduction." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1240.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Medicine (Pharmacology)." Discipline: Pharmacology; Department/School: Medicine.

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Cole, David K. "Biophysical and structural insights into the mechanism of T cell surface recognition." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442837.

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Cox, Georgina. "The FusB family of fusidic acid resistance proteins : structural and mechanistic insights." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.581880.

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The primary mechanism of resistance to the antibiotic fusidic acid (FA) in clinical strains of Staphylococcus aureus is expression of a FusB-type protein (usually FusB or FusC). These proteins bind elongation factor G (EF-G), the target of FA, and protect bacterial translation from FA-mediated inhibition. However, the interaction of these proteins with EF-G is poorly characterised, their structure has not been elucidated, and it is unknown how protection from FA is mediated. This thesis reports the first structure of a FusB-type protein, and begins to define the interaction between these proteins and EF-G. The 3D structure of FusC reveals a monomer composed of two distinct domains. The N-terminal domain comprises a four-helix bundle exhibiting similarity to existing protein structures. By contrast, the C-terminal domain forms a novel fold of helices and B-sheet with four conserved cysteine residues coordinating a central zinc ion. This region of FusB-type proteins was found to mediate a high-affinity interaction with the C-terminal domains of EF-G in vitro, yielding a complex with a 1: 1 stoichiometry. The protein-protein interaction occurs away from the FA binding site of EF-G, suggesting that FusB-type proteins do not mediate FA-resistance through direct steric hindrance of the interaction between FA and its target. Instead, FusB-type proteins interact with the portion of EF-G known to make contact with the inside of the ribosome. Owing to structural constraints, EF-G appears unable to simultaneously bind to FusB-type proteins and the ribosome. Based on these findings, a model of FusB-type resistance is proposed in which FusB-type proteins drive the release of frozen FA-EF-G-GDP complexes from the ribosome by competing for binding to EF-G.

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47

Ciniawsky, Susanne. "Structural and functional insights into the mechanism of the Pex1/6 complex." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-183979.

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Peroxisomes are highly dynamic organelles of eukaryotic cells, carrying out essential oxidative metabolic processes. These organelles scavenge reactive oxygen species such as hydrogen peroxide (H2O2) and catabolise fatty acids, which are particular hallmarks and highly conserved features of peroxisomes among different species. Peroxisomal proteins and enzymes are encoded by nuclear DNA and therefore, targeted post-translationally into the peroxisomal matrix. A special class of proteins, collectively called peroxins, perform certain cellular tasks, such as peroxisomal matrix protein import or membrane development in order to maintain peroxisome biogenesis as well as a constant flux of matrix proteins into peroxisomes. The type II AAA+ peroxins Pex1/Pex6 are a core component of the peroxisomal matrix protein import system. ATPases of the AAA+ family of proteins generally assemble into large, macromolecular machines, structurally remodelling their substrate protein, which is driven by the hydrolysis of ATP. The main function of Pex1/6 complexes is to release the receptor Pex5 from peroxisomal membranes after matrix protein import. This relocation of Pex5 into the cytosol ensures a constant pool of available receptor molecules for subsequent cycles of protein import into peroxisomes. Accordingly, certain mutations in mammalian Pex1/Pex6 proteins compromise peroxisome biogenesis and thus, lipid metabolism, causing severe genetic Zellweger diseases in humans. In collaboration with Professor Ralf Erdmann and colleagues at the Ruhr-Universität Bochum, we characterize the structure and function of the AAA+ Pex1/6 complex from yeast Saccharomyces cerevisiae. Single particle electron microscopy (EM) in combination with biochemical assays allows us to analyze how ATP turnover is related to the biological function of the Pex1/6 complex. This study presents EM structures of Pex1/6 complexes assembled in the presence of ADP, ATP, ADP-AlFx and ATPγS, providing a comprehensive structural characterization of the heterohexameric type II AAA+ complex in different nucleotide states. Our EM reconstructions reveal an unexpected triangular overall shape, different than observed for the closely related and well-characterized homohexameric AAA+ protein p97. We show that the heterohexameric Pex1/6 complex is composed of a trimer of heterodimers with alternating subunit arrangement of Pex1 and Pex6 moieties. Furthermore, our results suggest that conserved aromatic residues, lining the central pore of the Pex1/6 D2 ring mediate substrate interactions. These residues correspond to substrate interaction regions in related AAA+ proteins. Comparing Pex1/6 EM reconstructions in different nucleotide states implicates that the mechanical function of Pex1/6 involves an N- to C-terminal protein translocation mechanism along the central pore. The Pex1/6 EM structures resolve symmetric and asymmetric large-scale domain motions, which likely create a power stroke during cycles of ATP binding and hydrolysis. We conclude that Pex5 is probably partially or completely unfolded while it is threaded through the central pore of Pex1/6 complexes. In addition, ATP hydrolysis assays of Pex1/Pex6 complexes containing single amino acid exchanges in individual Walker B motifs reveal that not all active sites are functionally equivalent. In isolated complexes, ATP turnover mainly occurs in Pex6 D2 domains, while Pex1 subunits sustain the structural integrity of the complex. We further resolve the structures of Pex1/6 Walker B variants and observe mutually exclusive protomer-protomer communication. In the Pex1/6 complex, a Walker B mutation induces ATP hydrolysis in the adjacent D2 domain, presenting a structural framework of protomer-protomer communication in the AAA+ heterohexamer.

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48

Melo, Arthur [Verfasser]. "Structural insights into the activation mechanism of the EHD family / Arthur Melo." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/115300805X/34.

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49

La-Borde, Penelope Jane. "Activation of the vasopressin and oxytocin receptor family : structural and mechanistic insights." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7639/.

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G-protein-coupled receptors (GPCRs) are major pharmaceutical drug targets due to their crucial role in cell signalling. The neurohypophysial peptide hormones [Arg⁸] vasopressin (AVP) and oxytocin (OT) signal through a subfamily of GPCRs, comprising the AVP receptors (V₁a R, V₁bR and V₂R) and the OT receptor (OTR). The aim of this work was to understand the molecular basis of receptor activation by defining exact contacts between agonist and receptor. Molecular modelling suggested that two conserved negatively-charged residues may be important for agonist binding and receptor activation. Interactions between agonists and the human AVP/OT receptors were probed using a combination of site-directed mutagenesis of the receptors and ligands incorporating modifications at specific points. The wild-type (WT) and mutant receptors were expressed in HEK 293T cells and pharmacologically characterised with respect to ligand binding, receptor activation, cell surface expression and ligand-induced internalisation when stimulated by endogenous, or modified, ligands. Mutual exchange of functional groups revealed direct interactions between AVP/OT/[Arg⁸] vasotocin (AVT; a chimera of AVP and OT) and the human AVP/OT receptors required for receptor activation. These studies advance current understanding of the molecular basis of receptor activation and have the potential to facilitate future rational drug design.

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Pazicky, Samuel [Verfasser]. "Structural and functional insights into apicomplexan gliding and its regulation / Samuel Pazicky." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1226936415/34.

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