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Dalton, James Andrew Rupert. "The homology modelling of protein-ligand interactions." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515451.
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Asi, Azizah. "Homology modelling and simulations of the major facilitator superfamily transporters." Thesis, University of Oxford, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.724976.
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Jones, Martin Lionel. "Analysing loop selection criteria in homology modelling of proteins using an object-oriented database." Thesis, University of Aberdeen, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387153.
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One of the most difficult problems in modern biochemistry is that of accurately predicting a protein's three dimensional structure from its sequence (the protein folding problem). This structure is essential for a proper understanding of how a protein functions. As experimental derivation of a protein's structure is far more time consuming than deriving a protein's sequence, prediction of structure from sequence is an important goal for many protein biochemists; several methods have been suggested for this. Given a protein of known structure of similar sequence to the protein you wish to model homology modelling is the method most likely to produce a fairly good model. In this work a tool was produced for examining the various stages of homology modelling and analysing how well various method for carrying out these stages perform. The tool produced consists of an object-oriented database of protein structures and testbed software written in a mixture of PROLOG and DAPLEX. Tests were carried out using this software to examine the predictivity of various guidelines suggested in the literature for the loop selection stage of cut and paste homology modelling. The results of these tests produced surprising new information on the relative importance of different factors which may be used to choose between candidate fragments for the variable regions of a protein being modelled. The results of the application of these automated modelling methods were then compared with a short series of modelling tests using human modellers in an attempt to measure how the usual modelling procedures using 'hand and eye' compare with automated measures. Finally the results of the tests carried out were used to guide the production of a model of a previously unmodelled serine proteinase.
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Anye, Valentine. "Structural analysis of induced mutagenesis A’ protein from mycobacterium tuberculosis and of a thermophillic GH9 cellulase." University of the Western Cape, 2014. http://hdl.handle.net/11394/4320.
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Masters of ScienceThe three-dimensional structures of proteins are important in understanding their function and interaction with ligands and other proteins. In this work, the structures of two proteins, ImuA’ from mycobacterium tuberculosis and GH9 C1 cellulase from a metagenomic library, were analysed using structural biological and modelling techniques. The gene encoding ImuA’ was amplified by two-step PCR, cloned, and expressed in E. coli. The recombinant ImuA’ produced was found to be largely insoluble. The insoluble protein was successfully solubilized in 8M urea but refolding the protein to its native structure was unsuccessful. By homology modelling, a 3D model of ImuA’ was obtained from a partly homologous protein RecA. In comparison to RecA, ImuA’ appears to lack some loop amino acids critical for DNA binding. Hence ImuA’ is postulated to not bind DNA. The second protein, GH9 C1 cellulase, was produced in E. coli. The protein was purified by chromatographic techniques and crystallized in a precipitant to protein ratio of 1:2 by hanging and sitting drop crystallization methods. The reservoir solution was made up of 15-30% (w/v) PEG 3350, 200 mM salt and 100 mM Tris-HCL pH 7.5-8.5. The protein crystals only diffracted x-rays to 4 å resolution which could not be used to obtain a crystal structure of the protein. The diffraction data, however, showed the crystal to be monoclinic with space group P2. Homology modelling revealed GH9 C1 cellulase to be a two domain protein with a smaller N-terminal Ig-like domain and a larger catalytic domain.The catalytic domain retains two ca2+ binding sites, which potentially stabilize the active site conformation and increase thermostability of the protein. Overall GH9 C1 cellulase is structurally similar to other GH9 cellulases, suggesting that its catalytic mechanism may be conserved.
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Nematollahi, Alireza. "Kynurenine Aminotransferases as Novel Targets in Neurodegenerative and Cognitive Disorders using Rational Drug Discovery." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15985.
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This dissertation is premised on observations that kynurenine aminotransferase (KAT) isozymes play a role in neurodegenerative and cognitive disorders and has considered the probable mechanisms of action of inhibitors. This thesis used rational drug discovery in the design of novel reversible human KAT-2 inhibitors. This work is presented in publication format with six chapters. Chapter 1 includes two published papers of which the first article is about the evolution of antipsychotic drugs used in the clinic to overcome both positive and negative symptoms of schizophrenia with an emphasis on the main pharmacophore features of these drugs, and the second article is a review of KATs and their existing inhibitors. Chapter 2 presents the structural features of KAT-3 with observations on its inhibition using molecular modeling and computational studies. Chapter 3 investigates the expression, purification and crystallization of human KAT-2. Chapter 4 considers the crystal structure of human KAT-2 and properties of the active site of this enzyme, which relates to the mechanism of action of the KAT isozymes. Chapter 5 presents the design and synthesis of a novel reversible inhibitor of human KAT-2, a promising lead as assessed by an HPLC-based bioassay, as well as a study of the binding affinity by the surface plasmon resonance technique to probe further its probable mechanism of action. The final chapter, Chapter 6, summarizes the results of this research with suggestions for possible future research directions.
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Kroon, Matthys Christoffel. "High-throughput modelling and structural investigation of cysteine protease complexes with protein inhibitors." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1001619.
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The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb
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Pereira, Ana Catarina da Silva. "Structural investigation of the Bacillus subtilis morphogenic factor RodZ." Master's thesis, Faculdade de Ciências e Tecnologia, 2013. http://hdl.handle.net/10362/11072.
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A thesis to obtain a Master degree in
Structural and Functional BiochemistryRodZ is a protein widely conserved in bacteria and a core component of the morphogenic apparatus of the cell. It is known to be required for assembly of the bacterial actin homologue, MreB, that controls cell wall synthesis and cell shape. The domain organization of RodZ consists of a well-conserved N-terminal (RodZn) with helix-turn-helix motif (HTH), a conserved transmembrane domain, and a conserved C-terminal domain (RodZc). RodZn, located in the cytoplasm, has been shown to interact with MreB actin-homologue by x-ray studies in T. maritima. However, the structure of RodZn from gram-positive B. subtilis showed low homology with the published one from gram-negative T. maritima. Here we present the solution structure of RodZn from B. subtilis determined for the first time, by NMR spectroscopy. Compared to previous structural data obtained from the crystallized RodZn from T. maritima and more recently from S. aureus, several differences could be observed, namely the length of the alpha-helices and the presence of an extended coil. Interaction studies were preformed between RodZn domain and MreB from which no significant results could be extrapolated. Since HTH motif is frequently associated with DNA interaction, the involvement of RodZn in DNA organization is being investigated. At the same time, RodZc domain, which structure has never been reported, was subject of study. Bioinformatic, biophysical and biochemical methodologies were employed to study this domain. A model based in a pseudo-ab initio methodology was built, revealing an Ig-like fold. The Ig superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Therefore, RodZ is thought to be a protein that establishes a link between the inner side of the cell membrane and the outer side, promoting spatiotemporal coordination between peptidoglycan synthesis and cell division.
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Muhammad, Sayyed Auwn. "Probabilistic Modelling of Domain and Gene Evolution." Doctoral thesis, KTH, Beräkningsvetenskap och beräkningsteknik (CST), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-191352.
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Phylogenetic inference relies heavily on statistical models that have been extended and refined over the past years into complex hierarchical models to capture the intricacies of evolutionary processes. The wealth of information in the form of fully sequenced genomes has led to the development of methods that are used to reconstruct the gene and species evolutionary histories in greater and more accurate detail. However, genes are composed of evolutionary conserved sequence segments called domains, and domains can also be affected by duplications, losses, and bifurcations implied by gene or species evolution. This thesis proposes an extension of evolutionary models, such as duplication-loss, rate, and substitution, that have previously been used to model gene evolution, to model the domain evolution. In this thesis, I am proposing DomainDLRS: a comprehensive, hierarchical Bayesian method, based on the DLRS model by Åkerborg et al., 2009, that models domain evolution as occurring inside the gene and species tree. The method incorporates a birth-death process to model the domain duplications and losses along with a domain sequence evolution model with a relaxed molecular clock assumption. The method employs a variant of Markov Chain Monte Carlo technique called, Grouped Independence Metropolis-Hastings for the estimation of posterior distribution over domain and gene trees. By using this method, we performed analyses of Zinc-Finger and PRDM9 gene families, which provides an interesting insight of domain evolution. Finally, a synteny-aware approach for gene homology inference, called GenFamClust, is proposed that uses similarity and gene neighbourhood conservation to improve the homology inference. We evaluated the accuracy of our method on synthetic and two biological datasets consisting of Eukaryotes and Fungal species. Our results show that the use of synteny with similarity is providing a significant improvement in homology inference.QC 20160904
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Fischer, Juliane [Verfasser], Birgit [Akademischer Betreuer] Dräger, Wolfgang [Akademischer Betreuer] Brandt, and Dietrich [Akademischer Betreuer] Ober. "Homology modelling, virtual screening and evolutionary analyses of plant enzymes metabolising putrescine / Juliane Fischer. Betreuer: Birgit Dräger ; Wolfgang Brandt ; Dietrich Ober." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2015. http://d-nb.info/1077768095/34.
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Riemann, Ralph Nico [Verfasser]. "Development of potential scaling methods to improve prediction of loops and binding interfaces at homology modelling and docking / Ralph Nico Riemann." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2008. http://d-nb.info/1034788132/34.
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Passetti, Fabio. "Implicações funcionais de eventos de splicing alternativo no proteoma humano." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-04092007-232516/.
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A pós-genômica surgiu como um próspero campo para que as infinidades de seqüências provenientes dos projetos genoma tenham os seus significados biológicos elucidados. Um dos mecanismos descritos na literatura capaz de gerar surpreendente diversidade protéica é o splicing alternativo (AS). Próximo de 22% das proteínas com estruturas tridimensionais resolvidas por difração de raios-X ou ressonância magnética nuclear (RMN) são humanas e pouco se sabe dos efeitos de eventos de splicing alternativo em suas funções. Uma vez que estas estruturas tridimensionais (3D) protéicas humanas são de alguma forma redundantes, o conjunto de genes humanos únicos que as correspondem é muito reduzido, em torno de 1%. Hoje em dia ainda são escassos os exemplos de duas isoformas de splicing alternativo de um mesmo gene com estruturas tridimensionais experimentais disponíveis. A variedade de proteínas que este evento pode potencialmente produzir é demasiado grande para que projetos de genômica estrutural em andamento consigam determinar suas estruturas. Isto tem inviabilizado, ainda que temporariamente, estudos sobre implicações funcionais de splicing alternativo no proteoma quando se utilizando dados estruturais experimentais. Entretanto, a bioinformática possibilita estudos deste porte com base nos dados de mapeamento no genoma, tanto de transcritos como de proteínas com estrutura tridimensional (3D) determinada. Torna-se possível, então, a prospecção de genes com isoformas de AS com estruturas 3D contendo informação adicional quando comparada à isoforma de AS. Produzimos para tal finalidade uma nova metodologia para detecção de eventos de AS no transcriptoma humano utilizando matrizes binárias para cada transcrito e estrutura de proteína 3D. Selecionadas as isoformas protéicas putativas, foram construídas 73 estruturas 3D utilizando conceitos de modelagem molecular por homologia. Foram escolhidas aleatoriamente 21 isoformas de AS para simulações por dinâmicas moleculares (SDM), e que cerca de 80% destes modelos se apresentaram estruturalmente estáveis. A anotação biológica relativa a cada fragmento não inserido na seqüência da proteína devido à sua remoção no mRNA resultante do evento de AS foi obtida e mostrou que mais de 80% delas possuem algum tipo de relevância funcional para a proteína. Concluímos que, para o nosso conjunto de dados, os eventos de splicing alternativo produzem isoformas que podem atuar como dominantes negativas, antagonistas ou atenuadoras da sua atividade biológica.The post-genomic era has emerged as one prosper field to deal with the huge amount of sequences produced by genome projects and increase the understanding of its biological meaning. One of the most surprising mechanisms capable to generate a lot of protein diversity is alternative splicing in immature mRNAs. No more than 22% of the known protein structures elucidated by X-ray diffraction or nuclear magnetic resonance (NMR) were made using human proteins and the knowledge about alternative splicing functional implications is weak. Since those human protein three-dimensional structures (3D) are redundant, the unique number of human genes represented by them is estimated around 1%. Nowadays there are only a few cases describing two isoforms that have their own protein 3D structures done experimentally. The variety that alternative splicing can produce is large enough to structural genome projects undergoing could determinate its structures, fact that have negating, at least for a while, large-scale studies about functional implications of alternative splicing using experimental data. However, bioinformatics turn possible this kind of projects using the mapping onto the genome of transcripts and the sequence of the known protein 3D structures. Using this approach we searched for alternative splicing isoforms which have at least one known protein structure with additional biological information when compared against the isoform. We have produced a new methodology for detecting alternative splicing in the human transcriptoma using binary matrices for each transcript and known 3D protein structure. After the selection of putative isoforms, there were constructed 73 3D protein using concepts of molecular modelling by homology. There were randomly selected 21 of them to the submitted to molecular dynamics simulations and 80% of them showed that they were structurally stable. The biological annotation of each non-inserted fragment due to alternative splicing shows that 80% of them have in some degree functional importance. Then, we conclude that, for our dataset, the alternative splicing events produce isoforms that can act as negative dominants, antagonists or even regulators of their biological activity.
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Born, Stephan. "Kartierung der Bindungstasche des humanen Bittergeschmacksrezeptors hTAS2R10." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2012/6139/.
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Die Bittergeschmacksrezeptoren stellen in der Superfamilie der G-Protein-gekoppelten Rezeptoren eine besondere Gruppe dar. Im Menschen können die 25 Rezeptoren eine große Anzahl unterschiedlichster Bittergeschmacksstoffe detektieren. Diese Substanzen können sowohl schädlich, wie etwa Strychnin, als auch der Gesundheit förderliche Arzneistoffe, wie etwa Chloramphenicol sein. Unter den Bittergeschmacksrezeptoren des Menschen gibt es eine Gruppe von drei Rezeptoren, die besonders viele Bitterstoffe detektieren können. Einer von ihnen ist der Rezeptor hTAS2R10.
In dieser Arbeit konnte sowohl experimentell als auch durch computergestützte Modellierung gezeigt werden, dass der hTAS2R10 nur eine Bindungstasche besitzt. Das stimmt mit den bisher ausführlich experimentell und in silico untersuchten Rezeptoren hTAS2R1, -R16, -R38 und -R46 überein. Die für die Agonisteninteraktionen nachweislich wichtigen Transmembrandomänen sind in den bisher untersuchten Bittergeschmacksrezeptoren, wie auch im hTAS2R10, die Transmembrandomänen 3, 5, 6 und 7. Die Untersuchungen zeigten, dass die Bindungstasche des hTAS2R10 in der oberen Hälfte des zum extrazellulären Raum gerichteten Bereichs lokalisiert ist. Insbesondere konnte für die untersuchten Agonisten Strychnin, Parthenolid und Denatoniumbenzoat gezeigt werden, dass die Seitenketten der Aminosäuren in Position 3.29 und 5.40 ausgeprägte agonistenselektive Wechselwirkungen eingehen. Weitere Untersuchungen haben ergeben, dass das weitgefächerte Agonistenspektrum des hTAS2R10 zu Lasten der Sensitivität für einzelne Bitterstoffe geht. Der Vergleich wichtiger Positionen im hTAS2R10, hTAS2R46 und mTas2r105 hat deutlich gemacht, dass sich die Bindungsmodi zwischen diesen Rezeptoren unterscheiden. Dies deutet auf eine getrennte evolutionäre Entwicklung der Bindungseigenschaften dieser Rezeptoren hin. Gleichfalls zeigten die Untersuchungen, dass einige Positionen wie z.B. 7.39 die Funktion aller untersuchten Bittergeschmacksrezeptoren prägen, sich jedoch die genaue Bedeutung im jeweiligen Rezeptor unterscheiden kann. Einzelne dieser Positionen konnten auch bei der Agonisteninteraktion des Rhodopsins und des β2-adrenergen Rezeptors beobachtet werden. Die Ergebnisse dieser Arbeit helfen dabei die Wechselwirkungen zwischen Bitterstoffen und den Bittergeschmacksrezeptoren zu verstehen und geben erste Einblicke in die Entwicklung der Rezeptoren in Hinblick auf ihren Funktionsmechanismus. Diese Erkenntnisse können genutzt werden, um Inhibitoren zu entwickeln, die sowohl ein wichtiges Werkzeug in der Rezeptoranalytik wären, als auch dazu genutzt werden könnten, den unerwünschten bitteren Geschmack von Medikamenten oder gesundheitsfördernden sekundären Pflanzenstoffen zu mindern. Damit könnte ein Beitrag zur Gesundheit der Menschen geleistet werden.In the Superfamily of G protein-coupled receptors the bitter taste receptors form a notable group. The 25 human receptors are able to detect a large group of structurally diverse bitter compounds. These compounds can be toxic – like strychnine – or have beneficial effects on health – like the pharmacological agent chloramphenicol. Three of these bitter taste receptors show a strikingly broad agonist spectrum. One of them is the hTAS2R10. It was shown empirically and by computational modelling that the hTAS2R10 has only one binding pocket. This agrees with the findings of studies on the bitter taste receptors hTAS2R1, -R16, -R38 and -R46. The domains important for agonist interaction in these receptors, as well as in the hTAS2R10, are the transmembrane domains 3, 5, 6 and 7. The results of this thesis show that the binding pocket of the hTAS210 is located in the upper part of the receptor which points into the direction of the extracellular area. Interestingly, it has been shown for the amino acid side chains in the positions 3.29 and 5.40, that they can interact with the analysed agonists strychnine, parthenolide and denatonium benzoate in an agonist-selective way. Further analyses showed that the broad tuning of the hTAS2R10 goes at the expense of the sensitivity to single agonists. The comparison of crucial positions in the hTAS2R10, hTAS2R46 and the mTas2r105 reveal that these receptors differ in their binding mode. These could be evidence that the binding abilities of these receptors evolved independently. However, the results show that some positions, e.g. 7.39, influence the receptor activity in all analysed receptors, but the function of these positions in the receptors could be different. Some of these positions also have an influence on the agonist-receptor interaction of Rhodopsin and the β2-adrenergic receptor. The findings in this thesis contribute to the knowledge about interaction between bitter receptors and bitter compounds. The results also provide insight into the evolvement of receptor functions. These outcomes can be of use for the development of inhibitors which could serve as analytical tools in taste research. Furthermore, such inhibitors could be used to reduce the bitter taste of medicine and healthy plant compounds and thus increase palatability. This could contribute to improve human well-being.
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Afzelius, Lovisa. "Computational Modelling of Structures and Ligands of CYP2C9." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4016.
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BORDOGNA, ANNALISA. "Predicting the binding modes of protein complexes: new strategies for molecular docking." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/19617.
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Docking approaches and homology modelling procedures have recently become protagonists in the structural prediction of protein complexes. Therefore, in the last years it raised the need of developing more and more reliable tools and defining some guidelines to obtain accurate prediction of those structures. These goals are particularly relevant when only poorly accurate information about the proteins or the interaction (e.g. no interface indication for protein-protein docking, or protein models instead of experimental structures) is available.
In this thesis several strategies based on combining different computational techniques are proposed to overcome the limitations of the sampling stage of ligand- and protein-protein docking approaches and to broaden the possibility of predicting the structure of protein complexes. In particular, in the field of protein-protein docking algorithms, the combination of two existing docking methods (HADDOCK and ZDOCK) was proposed, allowing to obtain a method (called ZADDOCK) able to overcome the limitations of the single strategies used and to sum their strengths. Moreover, both for ligand- and protein-protein docking, an analysis of the relationships between docking results and the quality of homology models was performed to assess the possibility of an a priori prediction of the accuracy of docking results on the basis of the evaluation of model quality indices.
The results obtained for ZADDOCK show on average a very good performance of the method, that produced reliable predictions without the need of any interface data to guide the sampling step. This allows its employment in the study of complexes for which no experimental information is available and bioinformatics interface prediction fails. Moreover, an accurate description of the intermolecular interactions occurring in protein complexes was obtained, which is a key information to drive subsequent experimental work. The quality of ZADDOCK results indicates that the strategy of combining different computational techniques is a very promising avenue for the development of new docking approaches.
As for the use of homology models in docking calculations, in this work it is demonstrated the possibility of a priori predicting the accuracy of ligand-protein docking results on the basis of model quality indices, with the development of a strategy based on the comparison with homologous proteins. Moreover, the results found for protein-protein docking give the bases for a future development of a general prediction strategy of docking accuracy on protein models.
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Sousa, Bruno Gomes de. "Biodegrada??o de hidrocarbonetos arom?ticos polic?clicos: prospec??o metagen?mica e modelagem computacional 3-D de prote?nas." Universidade Federal do Rio Grande do Norte, 2011. http://repositorio.ufrn.br:8080/jspui/handle/123456789/13068.
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Previous issue date: 2011-05-23Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
Knowledge of the native prokaryotes in hazardous locations favors the application of biotechnology for bioremediation. Independent strategies for cultivation and metagenomics contribute to further microbiological knowledge, enabling studies with non-cultivable about the "native microbiological status and its potential role in bioremediation, for example, of polycyclic aromatic hydrocarbons (HPA's). Considering the biome mangrove interface fragile and critical bordering the ocean, this study characterizes the native microbiota mangrove potential biodegradability of HPA's using a biomarker for molecular detection and assessment of bacterial diversity by PCR in areas under the influence of oil companies in the Basin Petroleum Geology Potiguar (BPP). We chose PcaF, a metabolic enzyme, to be the molecular biomarker in a PCR-DGGE detection of prokaryotes that degrade HPA s. The PCR-DGGE fingerprints obtained from Paracuru-CE, Fortim-CE and Areia Branca-RN samples revealed the occurrence of fluctuations of microbial communities according to the sampling periods and in response to the impact of oil. In the analysis of microbial communities interference of the oil industry, in Areia Branca-RN and Paracuru-CE was observed that oil is a determinant of microbial diversity. Fortim-CE probably has no direct influence with the oil activity. In order to obtain data for better understanding the transport and biodegradation of HPA's, there were conducted in silico studies with modeling and simulation from obtaining 3-D models of proteins involved in the degradation of phenanthrene in the transport of HPA's and also getting the 3-D model of the enzyme PcaF used as molecular marker in this study. Were realized docking studies with substrates and products to a better understanding about the transport mechanism and catalysis of HPA s
O conhecimento sobre os procariotos nativos em locais de risco favorece a aplica??o de biotecnologias para biorremedia??o. Estrat?gias independentes de cultivo, como metagen?mica, contribuem para aprofundar o conhecimento microbiol?gico, possibilitando estudos com organismos n?o cultiv?veis acerca do status microbiol?gico nativo e seu potencial papel na biodegrada??o de, por exemplo, Hidrocarbonetos Arom?ticos Polic?clicos (HAP s). Considerando o bioma de mangue uma interface fr?gil e cr?tica de fronteira com o oceano, este trabalho caracteriza a microbiota nativa de mangue com potencial biodegradador de HAP s utilizando um biomarcador molecular para detec??o e avalia??o da diversidade bacteriana em ?reas sob influ?ncia de ind?strias petrol?feras atrav?s da PCR-DGGE na Bacia Petrol?fera Potiguar (BPP). Foi escolhido um biomarcador molecular metab?lico, enzima PcaF, para detec??o de procariotos degradadores de HAP s. Com o biomarcador, fingerprints foram obtidos de amostras de Paracuru-CE, Fortim-CE e Areia Branca-RN, revelando a ocorr?ncia de flutua??es das comunidades microbianas de acordo com os per?odos de amostragem e em resposta ao impacto por petr?leo. Atrav?s da an?lise das comunidades microbianas frente ? interfer?ncia da ind?stria do petr?leo, em Areia Branca-RN e Paracuru-CE foi observado que o petr?leo ? determinante para a diversidade microbiana. Fortim-CE provavelmente n?o tem influ?ncia direta da atividade petrol?fera. No intuito de obter dados para o melhor entendimento do transporte e biodegrada??o de HAP s, foram desenvolvidos estudos in silico de modelagem e simula??o computacional a partir da obten??o de modelos 3-D de prote?nas envolvidas na degrada??o do fenantreno, no transporte de HAP s e tamb?m a obten??o do modelo 3-D da enzima PcaF. Estudos de dockings com substratos e produtos forneceram dados para o melhor entendimento sobre o mecanismo de transporte e cat?lise de HAP s
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Boukharta, Lars. "Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-212103.
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Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods. We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target. In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance. Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.
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Abdulganiyyu, Ibrahim A. "A single AKH neuropeptide activating three different fly AKH-receptors: an insecticide study via computational methods." Doctoral thesis, Faculty of Science, 2021. http://hdl.handle.net/11427/33621.
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Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies.
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Souleymane, Diallo. "Coding of tsetse repellents by olfactory sensory neurons: towards the improvement and the development of novel." University of the Western Cape, 2020. http://hdl.handle.net/11394/8236.
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Philosophiae Doctor - PhDTsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.
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Kaganjo, James Chege. "Structure-function studies of 5-aminolevulinic acid (ALA) synthases." Bowling Green State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1510583488812729.
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De, Vecchis Dario. "Gaining insights into mitochondrial membrane fusion through a structural and dynamic atomistic model of the mitofusin Fzo1p." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC001.
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Les mitochondries sont des organites dynamiques dont la morphologie dépend de l’équilibre fusion/fission de leurs membranes. Ce processus essentiel à la survie cellulaire est nommé dynamique mitochondriale et sa dérégulation est associée à des troubles neurologiques. Cependant les mécanismes précis régissant la dynamique mitochondriale ne sont pas élucidés. Cette thèse porte sur la protéine Fzo1p, une grande GTPase de la superfamille des Dynamin-related-Protein. C’est un élément clé impliqué dans la fusion mitochondriale de la membrane externe de la levure. Sa structure et sa dynamique ont été étudiées par modélisation et simulations de dynamiques moléculaires tout-atome dans une bicouche lipidique solvatée. Le modèle structural obtenu tient compte de données expérimentales, de template structuraux, et de modèles ab initio du domaine transmembranaire de Fzo1p. Ce modèle a été validé expérimentalement par mutagenèse dirigée. Des permutations de charges ont confirmé des ponts salins à longue distance prédits dans le modèle. En outre, des mutations ont montré que les domaines coiled-coil de Fzo1p, contrairement à sa partie N-terminale, sont indispensables à sa fonction. L’ensemble des résultats expérimentaux et in silico met en évidence l’implication des domaines charnières dans le changement conformationnel de Fzo1p, ainsi que des résidus critiques affectant sa stabilité. Les précisions atomiques obtenues sur l’interaction de Fzo1p avec le GDP permet de formuler des hypothèses sur le mécanisme moléculaire de la catalyse du GTP pour la fusion membranaire; voire à la compréhension de la dynamique mitochondrialeMitochondria are dynamic organelles whose morphology is determined by fusion and fission of their membranes. This essential process is known as mitochondrial dynamics. Defects in mitochondrial dynamics are associated with neurological disorders making the investigation of physiological relevance. However, the precise sequence of events that lead mitochondrial dynamics are still not well characterised. Fzo1p, a large GTPase of the Dynamin-Related Proteins superfamily, is a key component in mitochondrial outer membrane fusion in yeast. During this PhD project I built a model of the protein Fzo1p. The structure and dynamics of the model was investigated through molecular modelling and all-atom molecular dynamics simulation in a fully hydrated lipid bilayer environment. The Fzo1p structural model integrates information from several template structures, experimental knowledge, as well as ab initio models of the transmembrane segments. The model is validated experimentally through directed mutagenesis, for instance charge-swap mutations confirm predicted long-distance salt bridges. A series of mutants indicate that coiled-coil domains are required for protein function at variance with its N-terminal region. Overall, the experimental and in silico approaches pinpoint the hinge domains involved in the putative conformational change and identifies critical residues affecting protein stability. Finally, key Fzo1p-GDP interactions provide insights about the molecular mechanism of membrane fusion catalysis. The model provides insight on atomic level and proposes a structure that will be instructional to understanding mitochondrial membrane fusion
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Svangård, Erika. "Cytotoxic Cyclotides : Structure, Activity, and Mode of Action." Doctoral thesis, Uppsala universitet, Institutionen för läkemedelskemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6028.
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Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines. Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules. The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity. The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5. The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.
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Hill, Jamie Richard. "Fold recognition and alignment in the 'twilight zone'." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:353a9832-b2a4-41fb-a9f2-f3cae1a30039.
Full textAbstract:
At present, the most accurate approach to predicting protein structure, comparative modelling, builds a model of a target sequence using known protein structures as templates. Comparative modelling becomes markedly less accurate in the ‘twilight zone’, where the target protein shares little sequence identity with all known templates. There are two main causes of this inaccuracy: first, it becomes difficult to identify good structural templates; second, it becomes difficult to determine which amino acids in the template are structurally equivalent to those in the target. These are problems of fold recognition and target-template alignment respectively. In this thesis, new approaches are developed to address both these problems. The alignment problem is investigated in the special case of membrane proteins. These are key modelling targets as they resist structure determination and are pharmaceutically important. The approach taken here is to use ‘environment specific substitution tables’ (ESSTs)– that is, to alter the alignment scoring system for each local environment of the template structure. We show how ESSTs can be made for membrane proteins, tested for robustness of construction, and used to infer the most important evolutionary pressures acting on protein structure. The incorporation of ESSTs into a multiple sequence alignment method leads to more accurate alignments of membrane proteins, and so to more accurate models. Recently, algorithms have been developed that predict contacts in protein structures from a multiple sequence alignment of homologous sequences. We explore the potential of these predictions for fold recognition by developing an algorithm that makes no use of amino acid identity, and so should be agnostic to the existence of a ‘twilight zone’. We show that whilst this is not the case, our method is complementary to state-of-the-art approaches.
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Schmidt, Matthias Rene. "K+ channels : gating mechanisms and lipid interactions." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:51dc4149-d943-4dcd-bf5b-f04130456d84.
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Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K+ channels. Molecular models were built of the inwardly rectifying K+ channel Kir2.2, the bacterial homolog K+ channel KirBac3.1, and the twin pore (K2P) K+ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K+ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K+ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K+ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.
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Stahl, Gunther. "Entwicklung von Homologie-Modellen der Cytochrome P450 2A5 und 2A6." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964988984.
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Asses, Yasmine. "Conception par modélisation et criblage in silico d'inhibiteurs du récepteur c-Met." Phd thesis, Université Henri Poincaré - Nancy I, 2011. http://tel.archives-ouvertes.fr/tel-00653609.
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L'enjeu des travaux effectués au cours de cette thèse est l'extraction in silico de molécules potentiellement intéressantes dans le processus d'inhibition du récepteur tyrosine kinase c-Met. La faculté de cette protéine à interagir dans les phénomènes d'embryogenèse et de réparation tissulaires rendent son inhibition cruciale dans les traitements contre les développements tumoraux où c-Met se trouve impliquée. Dans ce but, la stratégie que nous avons employée implique l'utilisation de plusieurs méthodes in silico de conception rationnelle de médicaments. Nous avons utilisé comme support les multiples structures cristallographiques publiées sur la ProteinData Base (PDB). Un travail de modélisation par homologie fut tout d'abord nécessaire pour combler les lacunes des structures cristallographiques collectées. Afin d'échantillonner au mieux l'espace conformationnel du récepteur kinase c-Met et de caractériser sa flexibilité, une longue campagne de simulation de Dynamique Moléculaire (DM) fut menée concernant les formes apo et holo des structures cristallographiques disponibles. Pour compléter ces simulations, une partie du travail consista à utiliser également la méthode des modes normaux de vibration (NM). De ces 2 approches (DM et NM), nous avons extrait un ensemble de 10 conformères considérés comme les plus représentatifs de l'espace conformationnel simulé pour la kinase c-Met et avons proposé un mode de fonctionnement de ce récepteur. Utilisant les conformations extraites de l'échantillonnage conformationnel, nous avons ensuite mené une importante campagne de criblage virtuel sur plusieurs chimiothèques constituant au total environ 70.000 composés. L'analyse des résultats de l'arrimage moléculaire nous a conduits à la sélection de plusieurs molécules intéressantes possédant théoriquement une bonne affinité pour la kinase c-Met. Ces molécules ont été soumises aux tests expérimentaux effectués par l'équipe de biologistes associée à nos travaux.
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THAMATAN, SRIKANTH GOUD. "HOMOLOGY MODELLING AND DOCKING STUDIES OF Bcl2L 10-HUMAN INVOLVING CANCER." Thesis, 2012. http://dspace.dtu.ac.in:8080/jspui/handle/repository/14120.
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Cancer known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. Homology modeling and flexible docking of Bcl2L10 has been studied in silico approach. Blast result was found to have similarity with Bcl2L10 of 48% identity with 2KUA. With the aid of Molecular dynamics and Molecular simulations studies it was identified that the generated structure was reliable. Active site of Bcl2L10 was identified by CASTP. Large potential drugs were designed for identifying molecules that can likely bind to protein target of interest. This structure was used to identify better inhibitor using docking studies. The drug derivatives were docked to the Bcl2L10 structure into the active site. The different drug derivatives designed were used for docking with the generated structure, among the 100 derivatives designed, fifth derivative showed highest docking result. The drug derivatives were docked to the protein by hydrogen bonding interactions and these interactions play an important role in the binding studies. Our investigations may be helpful for further studies.
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Tsai, Yau-Wei, and 蔡耀緯. "Expression, Purification, 3-D Homology Modelling and Immunoanalysis of Glucosyltransferases in Streptococcus mutans." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/02164008115640759582.
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碩士國立臺灣大學
口腔生物科學研究所
88
Glucosyltransferases (GTFs) of the Streptococcus mutans are responsible for the dental plaque formation in human oral cariogenesis by synthesizing glucans through hydrolysis of sucrose. There are three different kinds of GTFs in S. mutans: GtfB and GtfC are of membrane-associated form and responsible for synthesizing water-insoluble glucan, whereas GtfD is secretion form and responsible for water-soluble glucan. Much experimental work has been carried out to indicate the functional roles of these enzymes. All the three GTFs show very high homology in amino acid sequence while revealing great difference in the aspect of functional analysis. However, in present stage, no direct structural information is available to explain such functional discrimination. Therefore we have proceeded this work to approach such problem by comparing various GTFs in the aspect of three-dimensional structural characterization. (A) Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, molecular modelling has been carried out to build up the 3-D models of GTFs and correlate the structure-functional interpretation in order to understand the catalytic behavior of GTFs. (B) Chia and coworkers have results suggested that monoclonal antibody recognizing a 19-residue fragment, Gtf-P1, can reduce the sucrase activity of GTFs to a large extent. We have accomplished the functional analyses in order to supplement structure-functional correlation on such inhibition mechanism. (C) With a longer viewing, only crystal structure can provide the complete direct structural evidence for molecular mechanism in atomic level. Therefore, the third part of our project is to proceed the crystallization of GTFs in order to obtain crystals prepared for the 3-D structural determination. 3-D models of all three GTFs in Streptococcus mutans have been accomplished. The results suggested that these enzymes retain a Tim-barrel structure constructing the active sites inside the barrels, which may provide the specificity pocket for the entrance of substrate sucrose, as well as the binding to enzymes and the catalysis. Gtf-P1 is located on the surface of the molecule and protrudes the N-terminal residues of its long a-helix into the vicinity of active site. This may allow the antibody binding resulting conformational change to affect the active site, and furthermore, reduce the activity. Our results have provided sufficient interpretation for such structure-functional correlation. However, more direct evidence to describe the reaction mechanisms is anticipated through the forthcoming crystal structural determination.
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Accardi, Michael. "Molecular characterization of the binding site of nematode GABA-A receptors." Thesis, 2010. http://hdl.handle.net/10155/106.
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Haemonchus contortus is a parasitic nematode that is controlled in large part by
nematocidal drugs that target receptors of the parasitic nervous system. Hco-UNC-49 is a
nematode GABA receptor that has a relatively low overall sequence homology to
mammalian GABA receptors but is very similar to the UNC-49 receptor found in the free
living nematode Caenorhabditis elegans. However, the nematode receptors do exhibit
different sensitivities to GABA which may be linked to differences in the putative GABA
binding domains. Mutational analysis conducted in this study identified at least one
amino acid, positioned near the GABA binding domain, which may partially account for
differences in nematode GABA sensitivity. In addition, positions reported to be crucial
for GABA sensitivity in mammalian receptors also affect GABA sensitivity in Hco-
UNC-49 suggesting that the GABA binding domains of the mammalian and nematode
GABA receptors share some pharmacological similarities. However, there were some
differences observed. For example, in mammalian GABAA receptors amino acids from
both and subunits appear to be important for GABA sensitivity. For residues
examined in this study, only those on the UNC-49B subunit, and not UNC-49C, appear
important for GABA sensitivity.UOIT
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Riemann, Ralph Nico. "Development of potential scaling methods to improve prediction of loops and binding interfaces at homology modelling and docking /." 2005. http://www.jacobs-university.de/phd/files/1117028837.pdf.
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Mashkani, Baratali. "Small molecule inhibitors for type III receptor tyrosine kinases." Thesis, 2010. http://hdl.handle.net/1959.13/805670.
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Research Doctorate - Doctor of Philosophy (PhD)Colony stimulating Factor-1 Receptor (CSF-1R, FMS) and FMS-like Tyrosine Kinase-3 (FLT3) are members of the type III receptor tyrosine kinase (RTK) family. They have been implicated in a wide range of physiological and pathological processes including cancer and inflammatory diseases. Therefore blockade of their kinase activity using small molecule inhibitors (SMIs) may be a helpful treatment strategy for diseases associated with aberrant expression of FMS and FLT3. In this study, a cellular system for evaluation of SMIs was established by separate expression of human FMS and FLT3 in murine factor dependent FDC-P1 early myeloid cells. cDNAs encoding wild-type (WT) human FMS and FLT3 as well as leukaemia-associated constitutively active mutant forms of FLT3 (internal tandem duplication (ITD), D835V and D835Y) in the expression vector MSCV-IRES-GFP were introduced into FDC-P1 cells by retroviral transduction. Transduced cells were selected by Fluorescence-activated cell sorting (FACS) for green fluorescent protein GFP and growth in CSF-1 (also known as M-CSF), FLT3 ligand (FLT3L) or, in the case of FLT3 mutants, in the absence of growth factor. The coding regions for the CSF-1 and FLT3L were cloned from RNA extracted from K562, human erythroleukaemia cells and recombinant growth factors were produced in the yeast, Pichia pastoris. Several known SMIs of one or more Type III RTKs were evaluated for inhibition of FMS and FLT3 driven cell proliferation. Imatinib, dasatinib and sunitinib are potent inhibitors of c-KIT, while PKC412 and CEP701 are FLT3 inhibitors. The potency and selectivity of these SMIs were evaluated by inhibition of cell growth in presence of either mouse granulocyte macrophage colony-stimulating factor GM-CSF (control) or specific human growth factors (CSF-1 and FLT3L) and confirmed by inhibition of FMS and FLT3 phosphorylation upon stimulation by their cognate ligands. Each of these SMIs inhibited FMS kinase activity while FLT3 kinase (both WT and mutants) was inhibited by CEP701, PKC412 and to some degree by sunitinib, but not imatinib or dasatinib. The binding modes of the SMIs were predicted by molecular docking into homology models based on crystal structures of related kinases. Because kinase domains adopt different conformations in the inactive, active and inhibited states, multiple models of each kinase were evaluated. The binding mode data were correlated with selectivity and potency of the SMIs. Each of the small molecule inhibitors studied in this project represent a unique mode of activity against kinases, but in general they can be classified into three main categories. Firstly, molecules interacting mainly with the catalytic area (such as imatinib) taking advantage of the relatively unique substrate recognition site to be relatively selective, but affected adversely by the conformational switch during activation of the kinase domain. Secondly, molecules which interact exclusively with the ATP binding area (such as PKC412 and CEP701) can be effective on both active and inactive forms of kinases by taking advantage of binding to the area with least conformational changes during activation. However, it comes at the cost of less selectivity as this area is widely conserved among different types of kinases. Dasatinib, on the other hand, seems to have benefited from a kind of balanced interaction with both of these areas enabling it to be potent as well as relatively selective for the kinases with a threonine as gate-keeper residue. These examples show that extension of the purine-like core structure is required for high potency; otherwise the inhibitor (a molecule such as sunitinib) will not be able to compete with high concentration of ATP for binding to the active conformation of kinase. Extensions toward the ribose and phosphate groups (in molecules such as PKC412 and CEP701) result in increased potency, but decreased selectivity. To achieve higher potency and relative selectivity at the same time, the core structure should be extended toward the catalytic area (i.e. dasatinib). However, it should be limited to the vicinity of gate-keeper residue; otherwise the molecule will be vulnerable to the conformational changes during activation as explained for imatinib. The implications for design of SMIs of tyrosine kinases are discussed. Since the catalytic region is less stringently conserved and more influenced by conformational changes on activation, there is a high possibility of point mutations giving rise to resistance against SMIs targeting this region. If highly selective inhibitors are required, targeting of the catalytic area will be the choice, but if the aim is preventing or overcoming drug resistance in cancers due to mutations in the catalytic area (e.g. T670I in KIT) or strongly favouring the active conformation of the kinase domain (e.g. D816V in KIT or D835V/Y in FLT3), then the hinge region should be considered as the target area. It also will be possible to balance the selectivity and the potency by designing molecules that bridge the catalytic area and the hinge region. These findings will help in the design of new SMIs against the kinases according to each specific problem.
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Kaji, Mark. "A molecular characterization of agonists that bind to Hco-UNC-49, a GABA-gated chloride channel from Haemonchus contortus." Thesis, 2012. http://hdl.handle.net/10155/298.
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Haemonchus contortus is a blood feeding parasitic nematode infecting ruminants causing anemia and poor health at great economic cost. The ability to pharmaceutically control infection has been challenged by the rapid development and spread of drug resistance. The discovery of new targets is therefore required for sustainable parasite control. UNC-49 is a nematode ligand-gated ion channel that plays an important role in muscle contraction required for normal locomotion. However, little is known regarding its sensitivity to different agonists and how they interact with the binding site. This thesis describes an investigation into the efficacy of a range of classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus oocytes. The results of our electrophysiological recordings indicate that there is a size requirement for full agonism of the Hco-UNC-49 binding site. Furthermore, a number of molecules that are known to act on vertebrate GABA receptors have no effect on Hco-UNC-49. This suggests that the binding site of nematode GABA receptors does exhibit some unique properties. These findings could possibly be exploited to develop new drugs that specifically target GABA receptors from parasitic nematodes.UOIT
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Benedetti, G., SANTIS Pasquale DE, Stefano MOROSETTI, and Antonio PALLESCHI. "Analisi teorica delle relazioni struttura-funzione di catene di acidi ribonucleici: previsione delle strutture secondarie ottimali sulla base di criteri termodinamici, sperimentali e comparativi." Doctoral thesis, 1990. http://hdl.handle.net/11573/386795.
Full textAbstract:
Vengono analizzate le relazioni struttura-funzione che coinvolgono la struttura secondaria di una catena polinucleotidica di RNA.
La ricerca si è sviluppata in 3 fasi:a) sviluppo di un algoritmo di previsione teorica del folding secondario di RNA; b) previsione di un modello secondario per l'introne IVS rRNA della Tetrahymena thermophila e costruzione di una ipotesi di folding terziario a bassa risoluzione; c) analisi e sviluppo di un algoritmo di ricerca di motivi strutturali topologicamente equivalenti tra sequenze diverse di RNA, relazionate funzionalmente e quindi strutturalmente.
I risultati ottenuti mostrano come l'approccio teorico qui utilizzato può essere un valido strumento nello studio delle relazioni struttura-funzione di acidi ribonucleici e nella previsione di modelli strutturali in grado di interpretare più razionalmente il funzionamento di queste sofisticate macromolecole biologiche.
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Gowri, V. S. "Analysis Of Protein Evolution And Its Implications In Remote Homology Detection And Function Recognition." Thesis, 2007. https://etd.iisc.ac.in/handle/2005/568.
Full textAbstract:
One of the major outcomes of a genome sequencing project is the availability of amino acid sequences of all the proteins encoded in the genome of the organism concerned. However, most commonly, for a substantial proportion of the proteins encoded in the genome no information in function is available either from experimental studies or by inference on the basis of homology with a protein of known function. Even if the general function of a protein is known, the region of the protein corresponding to the function might be a domain and there may be additional regions of considerable length in the protein with no known function. In such cases the information on function is incomplete.
Lack of understanding of the repertoire of functions of proteins encoded in the genome limits the utility of the genomic data. While there are many experimental approaches available for deciphering functions of proteins at the genomic scale, bioinformatics approaches form a good early step in obtaining clues about functions of proteins at the genomic scale (Koonin et al, 1998). One of the common bioinformatics approaches is recognition of function by homology (Bork et al, 1994). If the evolutionary relationship between two proteins, one with known function and the other with unknown function, could be established it raises the possibility of common function and 3-D structure for these proteins(Bork and Gibson, 1996). While this approach is effective its utility is limited by the ability of the bioinformatics approach to identify related proteins when their evolutionary divergence is high leading to low amino acid sequence similarity which is typical of two unrelated proteins (Bork and Koonin, 1998). Use of 3-D structural information, obtained by predictive methods such as fold recognition, has offered approaches towards increasing the sensitivity of remote homology detection 9e.g., Kelley et al, 2000; Shi et al, 2001; Gough et al, 2001).
The work embodied in this thesis has the general objective of analysis of evolution of structural features and functions of families of proteins and design of new bioinformatics approaches for recognizing distantly related proteins and their applications. After an introductory chapter, a few chapters report analysis of functional and structural features of homologous protein domains. Further chapters report development and assessment of new remote homology detection approaches and applications to the proteins encoded in two protozoan organisms. A further chapter is presented on the analysis of proteins involved in methylglyoxal detoxification pathways in kinetoplastid organisms.
Chapter I of the thesis presents a brief introduction, based on the information available in the literature, to protein structures, classification, methods for structure comparison, popular methods for remote homology detection and homology-based methods for function annotation.
Chapter 2 describes the steps involved in the update and improvements made in this database. In addition to the update, the domain structural families are integrated with the homologous sequences from the sequence databases. Thus, every family in PALI is enriched with a substantial volume of sequence information from proteins with no known structural information.
Chapter 3 reports investigations on the inter-relationships between sequence, structure and functions of closely-related homologous enzyme domain families.
Chapter 4 describes the investigations on the unusual differences in the lengths of closely-related homologous protein domains, accommodation of additional lengths in protein 3-D structures and their functional implications.
Chapter 5 reports the development and assessment of a new approach for remote homology detection using dynamic multiple profiles of homologous protein domain families.
Chapter 6 describes development of another remote homology detection approach which are multiple, static profiles generated using the bonafide members of the family. A rigorous assessment of the approach and strategies for improving the detection of distant homologues using the multiple profile approach are discussed in this chapter.
Chapter 7 describes results of searches made in the database of multiple family profiles (MulPSSM database) in order to recognize the functions of hypothetical proteins encoded in two parasitic protozoa.
Chapter 8 describes the sequence and structural analyses of two glyoxalase pathway proteins from the kinetoplastid organism Leishmania donovani which causes Leishmaniases. An alternate enzyme, which would probably substitute the glyoxalase pathway enzymes in certain kinetoplastid organisms which lack the glyoxalase enzymes are also discussed.
Chapter 9 summarises the important findings from the various analyses discussed in this thesis.
Appendix describes an analysis on the correlation between a measure of hydrophobicity of amino acid residues aligned in a multiple sequence alignment and residue depth in 3-D structures of proteins.
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Gowri, V. S. "Analysis Of Protein Evolution And Its Implications In Remote Homology Detection And Function Recognition." Thesis, 2007. http://hdl.handle.net/2005/568.
Full textAbstract:
One of the major outcomes of a genome sequencing project is the availability of amino acid sequences of all the proteins encoded in the genome of the organism concerned. However, most commonly, for a substantial proportion of the proteins encoded in the genome no information in function is available either from experimental studies or by inference on the basis of homology with a protein of known function. Even if the general function of a protein is known, the region of the protein corresponding to the function might be a domain and there may be additional regions of considerable length in the protein with no known function. In such cases the information on function is incomplete.
Lack of understanding of the repertoire of functions of proteins encoded in the genome limits the utility of the genomic data. While there are many experimental approaches available for deciphering functions of proteins at the genomic scale, bioinformatics approaches form a good early step in obtaining clues about functions of proteins at the genomic scale (Koonin et al, 1998). One of the common bioinformatics approaches is recognition of function by homology (Bork et al, 1994). If the evolutionary relationship between two proteins, one with known function and the other with unknown function, could be established it raises the possibility of common function and 3-D structure for these proteins(Bork and Gibson, 1996). While this approach is effective its utility is limited by the ability of the bioinformatics approach to identify related proteins when their evolutionary divergence is high leading to low amino acid sequence similarity which is typical of two unrelated proteins (Bork and Koonin, 1998). Use of 3-D structural information, obtained by predictive methods such as fold recognition, has offered approaches towards increasing the sensitivity of remote homology detection 9e.g., Kelley et al, 2000; Shi et al, 2001; Gough et al, 2001).
The work embodied in this thesis has the general objective of analysis of evolution of structural features and functions of families of proteins and design of new bioinformatics approaches for recognizing distantly related proteins and their applications. After an introductory chapter, a few chapters report analysis of functional and structural features of homologous protein domains. Further chapters report development and assessment of new remote homology detection approaches and applications to the proteins encoded in two protozoan organisms. A further chapter is presented on the analysis of proteins involved in methylglyoxal detoxification pathways in kinetoplastid organisms.
Chapter I of the thesis presents a brief introduction, based on the information available in the literature, to protein structures, classification, methods for structure comparison, popular methods for remote homology detection and homology-based methods for function annotation.
Chapter 2 describes the steps involved in the update and improvements made in this database. In addition to the update, the domain structural families are integrated with the homologous sequences from the sequence databases. Thus, every family in PALI is enriched with a substantial volume of sequence information from proteins with no known structural information.
Chapter 3 reports investigations on the inter-relationships between sequence, structure and functions of closely-related homologous enzyme domain families.
Chapter 4 describes the investigations on the unusual differences in the lengths of closely-related homologous protein domains, accommodation of additional lengths in protein 3-D structures and their functional implications.
Chapter 5 reports the development and assessment of a new approach for remote homology detection using dynamic multiple profiles of homologous protein domain families.
Chapter 6 describes development of another remote homology detection approach which are multiple, static profiles generated using the bonafide members of the family. A rigorous assessment of the approach and strategies for improving the detection of distant homologues using the multiple profile approach are discussed in this chapter.
Chapter 7 describes results of searches made in the database of multiple family profiles (MulPSSM database) in order to recognize the functions of hypothetical proteins encoded in two parasitic protozoa.
Chapter 8 describes the sequence and structural analyses of two glyoxalase pathway proteins from the kinetoplastid organism Leishmania donovani which causes Leishmaniases. An alternate enzyme, which would probably substitute the glyoxalase pathway enzymes in certain kinetoplastid organisms which lack the glyoxalase enzymes are also discussed.
Chapter 9 summarises the important findings from the various analyses discussed in this thesis.
Appendix describes an analysis on the correlation between a measure of hydrophobicity of amino acid residues aligned in a multiple sequence alignment and residue depth in 3-D structures of proteins.
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Evers, Andreas [Verfasser]. "A new method for ligand-supported homology modelling of protein binding sites : development and application to the neurokinin-1 receptor / vorgelegt von Andreas Evers." 2004. http://d-nb.info/972859136/34.
Full text
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Akurugu, Wisdom Alemya. "Effects of nucleotide variation on the structure and function of human arylamine n-acetyltransferase 1." Thesis, 2012. http://hdl.handle.net/11394/3816.
Full textAbstract:
>Magister Scientiae - MScThe human arylamine N-acetyltransferase 1 (NAT1) is critical in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid and para-aminobenzoyl glutamate used in clinical therapy of tuberculosis (TB), as well as influencing the balance between detoxification and metabolic activation of these drugs. SNPs in this enzyme are continuously being detected and indicate inter-ethnic and inter-individual variation in the enzyme function. The effect of nsSNPs on the structure and function of proteins are routinely analyzed using SIFT and POLYPHEN-2 prediction algorithms. The false-negative rate of these two algorithms results in as much as 25% of nsSNPs. This study aimed to explore the use of homology modeling including residue interactions, Gibbs free energy change and solvent accessibility as additional evidence for predicting nsSNP effects on enzyme function.This study evaluated the functional effects of 14 nsSNPs identified in a South African mixed ancestry population of which 3 nsSNPs were previously identified in Caucasians. The SNPs were evaluated using structural analysis that included homology modeling, residue interactions, relative solvent accessibility,Gibbs free energy change and sequence conservation in addition to the routinely used nsSNP function prediction algorithms, SIFT and POLYPHEN-2. The structural analysis implemented in this study showed a loss of hydrogen bonds for S259R thereby affecting protein function which contradicts predictions obtained from SIFT and POLYPHEN-2 algorithms. The variant N245I was shown to be neutral but contradicted the predictions from SIFT and POLYPHEN-2. Structural analysis predicted that variant R242M would affect protein stability and therefore NAT1 function in agreement with POLYPHEN-2 predictions but contradicting predictions from SIFT. No structural changes were expected for variant E264K in agreement with predictions obtained from POLYPHEN-2 but contradicting results from SIFT. The functions of the remaining 10 nsSNPs were consistent with those predicted by SIFT and POLYPHEN-2 namely that four variants R117T, E167Q, T193S and T240S do not affect the NAT1 function whereas R166T, F202V, Q210P, D229H, V231G and V235A could affect the enzyme function.This study provided the first evaluation of the functional effects of 11 newly characterized nsSNPs on the NAT1 tuberculosis drug-metabolizing enzyme. The six functionally important nsSNPs predicted by all three methods and the four SNPs with contradictory results will be tested experimentally by creating a SNP construct that will be cloned into an expression vector. These combined computational and experimental studies will advance our understanding of NAT1 structure-function relationships and allow us to interpret the NAT1 genetic polymorphisms in individuals who are slow or fast acetylators. The results, albeit a small dataset demonstrate that the routinely used algorithms are not without flaws and that improvements in functional prediction of nsSNPs can be obtained by close scrutiny of the molecular interactions of wild type and variant amino acids.
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Silvério, Daniel José Vasconcelos. "Decoding partner specificity in opioid receptor family." Master's thesis, 2021. http://hdl.handle.net/10316/94337.
Full textAbstract:
Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e TecnologiaFoi realizada uma caracterização extensiva da família dos recetores de opióides para criar novo conhecimento acerca das propriedades farmacológicas e fisiológicas destes alvos de fármacos importantes. Foi realizado modulação por homologia usando estruturas do recetor opióide do tipo- κ (humano), do recetor de neurotensina 1 e recetor muscarínico M2 para gerar estruturas confiáveis de recetores de opióides ligados a proteína-G ou arrestina, cinco scores diferentes foram utilizados para selecionar o melhor modelo para cada caso. Foram realizadas simulações de Dinâmica Molecular (equilibração) de forma a relaxar os melhores modelos. As estruturas relaxas foram alinhadas com os modelos de parceiros para poder formar os complexos. Após a formação dos complexos foi aplicada uma ampla variedade de métodos computacionais para avaliar e providenciar uma descrição detalhada das interfaces de interação de todos os membros da família de recetores de opióides [µ (MOR), δ (DOR), κ (KOR), nociceptina (NOP) com os seus parceiros de ligação correspondentes (ARRs: ARR2, ARR3; proteína-G: Gi1, Gi2, Gi3, Go, Gob, Gz, Gq, G11, G12, G14, G15, Gs(sh), Gs(lo))]. Esta descrição inclui os seguintes parâmetros estruturais: distâncias inter-hélice, distâncias electroestáticas, resíduos que interagem, percentagens de interação dos resíduos, ligações de hidrogénio, pontes salinas, área de superfície acessível ao solvente, número de átomos à superfície e enterrados. Além disso, análise dinâmica, no âmbito da Análise de Modo Normal, foi também executada para avaliar dois parâmetros dinâmicos: mudanças de flexibilidade e mudanças no fold em flutuação média. A construção e análise destes 57 modelos envolvendo recetores de opióides representa uma nova e excitante análise de grandes dados dos determinantes da interface recetor de opióide-parceiros e constituí um passo seguinte na compreensão da especificidade funcional da família de recetores de opióides.
An extensive characterization of the opioid receptor family was carried out to create new knowledge about the physiological and pharmacological properties of these important drug targets. Homology modelling was performed using κ-type opioid receptor (human), neurotensin receptor 1 and muscarinic M2 receptor structures to generate reliable structures of complexes of opioid receptor bound to either G-protein or arrestin, five different scores were used to select the best model for each case. Molecular Dynamic simulations (equilibration) were performed in order to relax the best models. The relaxed structures were aligned with the partner models in order to form the complexes. After the complex formation a wide range of computational methods was applied to assess and provide a detailed description of the interaction interfaces of all members of the opioid receptor family [µ (MOR), δ (DOR), κ (KOR), nociceptin (NOP) with their corresponding binding partners (ARRs: ARR2, ARR3; G-protein: Gi1, Gi2, Gi3, Go, Gob, Gz, Gq, G11, G12, G14, G15, Gs(sh), Gs(lo))]. This description includes the following structural parameters: inter-helical distances, electrostatic distances, interacting residues, residue interaction percentages, hydrogen bonds, salt bridges, solvent accessible surface area, number of surface and buried atoms. Moreover, dynamic analysis, under the scope of Normal Mode Analysis, was also performed to evaluate two dynamical aspects of the complexes: flexibility changes and average fluctuation fold changes. The construction and analysis of these 57 models, involving opioid receptors, represents a novel and exciting big data analysis of opioid receptor-partners interface determinants and constitute a further step into the understanding of opioid receptor family functional specificity.
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Fatemi, Negah. "Structural and Functional Studies of ATP7B, the Copper(I)-transporting P-type ATPase Implicated in Wilson Disease." Thesis, 2011. http://hdl.handle.net/1807/31746.
Full textAbstract:
Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.
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Mulaudzi, Takalani. "Structural and functional characterisation of a novel signalling molecule in Arabidopsis thaliana." Thesis, 2011. http://hdl.handle.net/11394/3608.
Full textAbstract:
Philosophiae Doctor - PhDNitric Oxide (NO) influences a wide range of physiological processes in plants including growth and development, responses to abiotic and biotic stress and pathogen responses. NO binds to the heme group of the mammalian soluble guanylyl cyclase, which activates the enzyme to convert guanosine 5’ triphosphate (GTP) to a second messenger guanosine 3’, 5’ cyclic monophosphate (cGMP). Cyclic GMP further activates other signalling cascades including the regulation of protein kinases, ion gated channels and phosphodiesterases. In plants, a few GCs have been identified and these include AtGC1, AtBRI1, AtWAKL10, and AtPSKR1, however, a GC that contains a heme binding motif that senses NO has yet to be identified. In order to identify such molecules, a search motif based on conserved HNOX domains and the conserved and functionally assigned amino acid residues in the catalytic centres of annotated GCs was designed and used to search the Arabidopsis thaliana proteome. Several candidate molecules were identified including a flavin-containing monooxygenase (FMO)-like protein and the At5g57690 which is currently annotated as a diacylglycerol kinase. FMOs found in bacteria, yeast, and animals are the most important monooxygenases since they are involved in xenobiotic metabolism and variability in drug response. FMOs in plants are implicated in catalysing specific steps in auxin biosynthesis,metabolism of glucosinolates and pathogen defense mechanisms. The human diacylglycerol kinase acts as a lipid kinase that mediates a wide range of biological processes which include cell proliferation, differentiation and turmogenesis. In prokaryotes, the structure of Escherichia coli lipid kinase has been solved however, its function has not yet been demonstrated. So far, the occurrence of the diacylglycerol kinases in plants has not yet been reported, and their structure and function also remain elusive. The domain architecture of the 2 molecules (AtNOGC1 and At5g57690) identified by the HNOX-based search strategy revealed that these molecules contain a GC and a heme-binding motif that is conserved among all known heme-binding proteins.In this study, the role of AtNOGC1, a novel NO binding protein in higher plants was investigated and the results showed that this molecule contains an NO-dependant active GC domain. The sequence was first analysed and the location of the HNOX and the GC motifs highlighted. The protein was then recombinatly expressed as a His-SUMO fusion protein and the purification optimised by a second step of ion exchange chromatography. Electrochemical techniques such as cyclic voltammetry and square wave voltammetry were used to demonstrate the binding of NO and O2 to the AtNOGC1. Electrochemical data revealed that AtNOGC1 has a lower affinity for O2 and a higher affinity for NO, an important signalling molecule in plants.The presence of the GC activity in AtNOGC1 was investigated by conducting GC activity assays in vitro in the presence or absence of NO. The GC activity assays demonstrated that AtNOGC1 can synthesize cGMP from GTP in vitro. It was also noted that NO was required for the maximum activation of AtNOGC1 catalytic activity. NO-activated catalysis resulted in a >2 fold excess of cGMP production compared to an NO-independent GC activity assay. The effect of calcium in regulating the GC activity was also investigated and an increase in cGMP levels was observed however, this was just a preliminary finding that requires further experimentation.3 Homology models for both the FMO-like (AtNOGC1) and the diacylglycerol kinase(At5g57690) were built using Modeller program, and important amino acid residues underlying the heme-binding and GC motifs were identified. Residues corresponding to the motifs, which give signature to AtNOGC1 as an FMO, were also noted. In addition,computational functional prediction also suggested the role of AtNOGC1 in a number of processes which include ion binding and functioning as an FMO.Taken together, these findings suggest that AtNOGC1 is a novel Arabidopsis thaliana hemebinding protein that senses NO with higher affinity than for O2. Though AtNOGC1 is currently annotated as a FMO-like protein, it contains a NO-sensitive GC activity and shares limited sequence similarities with mammalian sGC and the recently identified HNOX domains. Homology modelling strongly suggests that AtNOGC1 and At5g57690 belong to the families of FMOs and diacylglycerol kinases respectively. The domain organisation of AtNOGC1 suggests that more of its functions still remain to be identified. The cloning and characterisation of the At5g57690 gene will provide possible means for further experimentation as well as affording more insights into the exact functions of lipid kinases in plants.
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Βουρεκάς, Αναστάσιος. "Μελέτες επί της δομής και της λειτουργίας του ριβονουκλεοπρωτεϊνικού συμπλόκου της RNase P από το Dictyostelium discoideum." 2007. http://nemertes.lis.upatras.gr/jspui/handle/10889/588.
Full textAbstract:
Η ριβονουκλεάση P είναι το ένζυμο το οποίο αναλαμβάνει την δημιουργία του 5´ ώριμου άκρου όλων των πρόδρομων μορίων tRNA. Πρόκειται για ένα ριβονουκλεο-πρωτεϊνικό σύμπλοκο το οποίο εντοπίζεται στα κύτταρα των οργανισμών και από τις τρεις κύριες φυλογενετικές περιοχές, τα Βακτήρια, τα Αρχαία και τους Ευκαρυώτες. Αποτελείται από μια υπομονάδα RNA απαραίτητη για την κατάλυση, ενώ το μέγεθος και ο αριθμός των πρωτεϊνικών υπομονάδων ποικίλλει από μια μικρή στα βακτήρια έως δέκα πρωτεΐνες στο ολοένζυμο που απομονώνεται από τα ανθρώπινα κύτταρα. Η υπομονάδες RNA των βακτηρίων καθώς επίσης και μερικών αρχαίων μπορούν να καταλύσουν την αντίδραση ωρίμανσης του tRNA απουσία της πρωτεΐνης in vitro, είναι δηλαδή ριβοένζυμα. Η ανακάλυψη αυτή διεύρυνε τις αντιλήψεις μας για τις ιδιότητες των βιομορίων και επανέφερε στο προσκήνιο την θεωρία του κόσμου του RNA. Στο ευκαρυωτικό ριβοένζυμο, ο ρόλος των πρωτεϊνών είναι πιο ουσιαστικός, καθώς η υπομονάδα RNA φαίνεται ότι χάνει μεγάλο μέρος της λειτουργικής της ανεξαρτησίας. Η διαλεύκανση των λειτουργών της κάθε υπομονάδας θα δώσει σημαντικές πληροφορίες για την εξέλιξη της RNase P από ένα αρχέγονο ένζυμο σε ένα πολύπλοκο ριβονουκλεοπρωτεϊνικό σύμπλοκο.
Η RNase P από το Dictyostelium discoideum διαθέτει μια απαραίτητη για την δραστικότητα υπομονάδα RNA όπως και όλα τα ένζυμα αυτού του είδους. Παράλληλα διαθέτει έντονο πρωτεϊνικό χαρακτήρα καθώς διαθέτει την χαμηλότερη πυκνότητα επιπολής σε σχέση με ένζυμα RNase P από άλλους οργανισμούς. Οι πληροφορίες αυτές προέρχονται από τον αρχικό χαρακτηρισμό του ενζυμικού συμπλόκου, και δεν παρέχουν στοιχεία για την ακριβή σύστασή του. Στην παρούσα μελέτη, πραγματοποιήθηκε κλωνοποίηση και χαρακτηρισμός ενός από τα γονίδια που εντοπίστηκαν στο γονιδίωμα του Dictyostelium, ομόλογα προς χαρακτηρισμένα γονίδια από τον άνθρωπο και άλλους ευκαρυώτες. Το γονίδιο drpp30 κωδικεύει μια πρωτεΐνη 40.7 kDa, σημαντικά μεγαλύτερη από τις ομόλογες της. Η πρωτεΐνη DRpp30 υπερεκφράστηκε σε βακτηριακά κύτταρα, και μετά τον χρωματογραφικό καθαρισμό της χρησιμοποιήθηκε για την παρασκευή πολυκλωνικών αντισωμάτων. Η συμμετοχή της DRpp30 στο μακρομοριακό σύμπλοκο της RNase P πιστοποιήθηκε με ανοσοβιοχημική προσέγγιση, ενώ η ανασυνδυασμένη πρωτεΐνη προσδένει τo pre-tRNA υπόστρωμα του ενζύμου, καθώς και την υπομονάδα RNA in vitrο. Το μοντέλο ομολογίας της DRpp30 βάσει της κρυσταλλικής δομής της ορθόλογης Ph1877 από τα αρχαία, φανερώνει ότι η πρωτεΐνη αποκτά τη δομή αβ βαρελιού (ΤΙΜ barrel fold).
Κατά τη διάρκεια της διατριβής, οι προσπάθειες για τον εντοπισμό του γονιδίου της RNA υπομονάδας ήταν σε εξέλιξη, όταν το εν λόγω γονίδιο αναγνωρίστηκε μέσω φυλογενετικών συγκρίσεων από την ομάδα του Norman Pace. Το μετάγραφο του γονιδίου εντοπίστηκε σε ενεργά κλάσματα RNase P, και παράλληλα εντοπίστηκε και ένα μικρότερο μετάγραφο του ίδιου γονιδίου. Προσδιορίστηκαν τα ακριβή 5´και 3´ άκρα των δύο αυτών μορίων και ακολούθησε κλωνοποίηση τους. Τα in vitro μετάγραφα των δύο κλωνοποιημένων αλληλουχιών μπορούν να υποκαθιστούν την ενδογενή RNA υπομονάδα του ολοενζύμου in vitro, ενώ δεν εντοπίστηκε έως τώρα ενζυμική δραστικότητα που να σχετίζεται με τα δύο αυτά μόρια.Ribonuclease P is a ubiquitus ribonucleoprotein enzyme, responsible for the production of the 5´ mature ends of all precursor tRNA molecules. RNase P endonucleolytic activity has been isolated from organisms representing the three domains of life, namely Bacteria, Archaea and Eukarya. It has been shown to contain an essential RNA subunit and one (Bacteria) or more (Archaea, Eukaryotes) proteins. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea have lost most of their functionality and require protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and structures for both RNA and protein subunits have emerged. The integration of structural and functional data is slowly forming a scenario for the evolution of RNase P from an ancient enzyme to a highly organized ribonucleoprotein complex. Dictyostelium discoideum RNase P harbors an essential RNA subunit, and has high protein content, as judged by its low boyant density. Nevertheless, our knowledge on the exact composition was limited. In the current study, a gene showing significant similarity to human Rpp30 RNase P protein subunit was identified in Dictyostelium genome. The gene encodes a protein (DRpp30) which is significantly larger than its homologues, due to an unusual C-terminus. The gene was cloned, overexpressed, and was used for the production of polyclonal antibodies. The participation of DRpp30 in the macromolecular complex of RNase P was verified by an immunobiochemical approach. The recombinant protein was shown to bind specifically both the RNase P RNA subunit and the pre-tRNA substrate in vitro, thus giving a first insight of its role in the holoenzyme complex. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold. While our efforts to isolate the gene encoding the RNA subunit of D. discoideum RNase P were in progress, Norman Pace and his group identified it through phylogenetic comparison. The full transcript of the gene was detected in active RNase P samples along with a smaller transcript of the same gene. The exact 5´and 3´ ends of both transcripts were identified and were cloned. Both these transcripts can substitute the endogenous RNA subunit in vitro, but no enzymatic activity associated with these RNA molecules could be detected so far.
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Serra, Patrícia Filipa Alves. "Viral surface glycoproteins as potential drug targets against HIV-1 and HIV-2 infections." Doctoral thesis, 2020. http://hdl.handle.net/10451/48510.
Full textAbstract:
AIDS (acquired immune deficiency syndrome) was discovered more than forty years ago, however, until now there is still no vaccine or cure for this disease. AIDS is caused by two human immunodeficiency virus (HIV) types, HIV-1 and HIV-2. HIV-1 is pandemic while HIV-2 is mainly confined to West Africa and western European countries (e. g., France, and Portugal). These two viruses share several common hallmarks, aside from a similar mode of transmission, similar intracellular replication mechanisms, and clinical consequences. In fact, as the disease progresses, people infected with HIV-2 will become vulnerable to the same spectrum of associated opportunistic infections and co-morbidities as people infected with HIV-1. Traditionally, HIV-2 infection is frequently neglected in global HIV/AIDS campaigns, however, a special emphasis must be given to HIV-2, which poses distinct challenges for prevention, diagnosis, and treatment to eradicate AIDS. None of the current drugs effectively prevents entry into the cells and the efficacy of the available drugs is very limited against HIV-2. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry. The discovery of multiple new hit compounds can be used as useful starting points towards drug candidates for HIV-1 and HIV-2 therapy. The viral gp120 and gp125 are critical for the recognition of receptors and allowing the internalization of viral content into the cell. Its modulation can lead to the disturbance of the entry viral mechanism.
In the absence of a complete crystallographic structure of HIV-2 envelope gp125 comprising variable domains, computer-aided modulation is crucial to identify structural features in the variable regions that correlate with HIV-2 tropism and susceptibility. Here, we developed and optimized a computer-assisted drug design approach of an important HIV-2 glycoprotein that allows us to explore and gain further insights at the molecular level into protein structures and interactions crucial for HIV-2 inhibition. A 3D structure of HIV-2ROD gp125 was generated by a homology modelling campaign. To disclose the importance of the main structural features and compare with experimental results, 3D-models of six mutants were also generated. These mutations revealed selectively impact the behaviour of the protein. Furthermore, molecular dynamics simulations were performed to optimize the models, and the dynamic behavior was tackled into account for structure flexibility and interactions network formation. It is primordial to understand the structural behaviour of these domains inserted in the main structure. Structurally, the mutations studied lead to a loss of aromatic features, very important for the establishment of π-π interactions, which could induce a structural preference by a specific coreceptor. Additionally, a drug discovery protocol to identify small molecules that inhibit the attachment and binding of HIV with host cells mediated by the envelope surface glycoproteins was developed. The discovery protocol based on molecular docking and virtual screening was developed to gp120 of HIV-1 to disclose molecular characteristics of potential new inhibitors of the viral attachment that we want to correlate with the homologous modulated protein of HIV-2.
Altogether, these findings reveal important aspects of the structural characterization of the glycoproteins fundamental to the viral entry mechanism of HIV. The computational approach offers numerous advantages to obtain these new insights into the structure-function relationship, and this translational methodology can lead to the improvement of the knowledge of viral glycoproteins and its application towards the battle against the HIV infection.
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Hang, Pei Chun. "Investigations into Streptomyces azureus Thiostrepton-resistance rRNA Methyltransferase and its Cognate Antibiotic." Thesis, 2008. http://hdl.handle.net/10012/4143.
Full textAbstract:
Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.
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(9143657), Phillip S. Rushton. "Structure of the Plant-Conserved Region of Cellulose Synthase and Its Interactions with the Catalytic Core." Thesis, 2020.
Find full textAbstract:
The processive plant cellulose synthase (CESA) synthesizes (1→4)-β-D-glucans. CESAs assemble into a six-fold symmetrical cellulose synthase complex (CSC), with an unknown symmetry and number of CESA isomers. The CSC synthesizes a cellulose microfibril as the fundamental scaffolding unit of the plant cell wall. CESAs are approximately 110 kDa glycosyltransferases with an N-terminal RING-type zinc finger domain (ZnF), seven transmembrane α-helices (TMHs) and a cytoplasmic catalytic domain (CatD). In the CatD, the uridine diphosphate glucose (UDP-Glc) substrate is synthesized into (1→4)-β-D-glucans. The ZnF is likely to facilitate dimers in the CSC. Recombinant class-specific region (CSR), a plant specific insertion to the C-terminal end of the CatD is also known to form dimers in vitro. The CSR sequence is the primary source of distinction between CESA isoforms and class structure. Also within the CESA CatD is a 125-amino acid insertion known as the plant-conserved region (P-CR), whose molecular structure was unknown. The function of the P-CR is still unclear, especially in the context of complete CESA and CSC structures. Thus, one major knowledge gap is understanding how multimeric CSCs synthesize multiple chains of (1→4)-β-D-glucans that coalesce to form microfibrils. The specific number of CESAs in a CSC and how interactions of individual CESA isoforms contribute to the CSC are not known. Elucidating the structure-function relationships of the P-CR domain, and with the consideration of the ability of CSR and ZnF domains to dimerize, it is possible to more completely model the structure of the CSC.
Recombinantly expressed rice (Oryza sativa) secondary cell wall OsCESA8 P-CR domain purifies as a monomer and shows distinct α-helical secondary structure by circular dichroism analysis. A molecular envelope of the P-CR was derived by small angle X-ray scattering (SAXS). The P-CR was crystallized and structure solved to 2.4 Å resolution revealing an anti-parallel coiled-coiled domain. Connecting the coiled-coil α-helices is an ordered loop that bends back towards the coiled-coils. The P-CR crystal structure fits the molecular envelope derived by SAXS, which in turn fits into the CatD molecular envelope. The best fit places the P-CR between the membrane and substrate entry portal. In depth analysis of structural similarity to other proteins, and 3D-surface structure of the P-CR, leads to hypotheses that it could function in protein-protein interactions as a dimer, trimer or tetramer in the CSC, that it could form protein-protein interactions with CESA-interacting proteins, and/or modulate substrate entry through its N- and/or C-terminus. From modeling, hypothetically important residues within the P-CR or related to the P-CR through potential protein contacts were mutated in Arabidopsis thaliana AtCESA1 constructs. These constructs were expressed in the temperature-sensitive radial swelling (rsw) rsw1-1 mutant of AtCESA1 to test for complementation of growth phenotypes at restrictive temperatures. Preliminary experiments indicate that some mutated CESA1 sequences fail to complement the rsw1-1 phenotype, suggesting that specific functions of individual amino can be tested using this system.
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Brunet, Jean-Frédéric. "Rôle des facteurs physico-chimiques du micro-environnement intestinal et des boucles inter-hélicales du Domaine I dans l’activité de la toxine insecticide Cry9Ca du bacille de Thuringe." Thèse, 2009. http://hdl.handle.net/1866/4183.
Full textAbstract:
Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe.Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.
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Briot, Julie. "Identification biochimique et fonctionnelle des domaines structuraux d’une sous-unité des canaux calciques." Thèse, 2018. http://hdl.handle.net/1866/21202.
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Stahl, Gunther [Verfasser]. "Entwicklung von Homologie-Modellen der Cytochrome P450 2A5 und 2A6 / vorgelegt von Gunther Stahl." 2002. http://d-nb.info/964988984/34.
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