Proteins are one of the most important molecules in biology. The wide range of functions of different proteins is also important for medical physics. Proteins are assembled by amino acids. These amino acids are connected by peptide bonds to form a protein. The function of a protein is decided by the composition and configuration of peptides, amino acids and their peptide bonds. Successful experiments with Xray Free-electron laser has lead to progress in structural biology, however there is still a need to crystallized samples in these experiments. In this project we have investigated three amino acids. These three amino acids are included in several protein that are hard to crystallize, glycine, valine and alanine. We have investigated their separate interatomic bonds by performing density functional calculations and evaluating the susceptibility of the bonds breaking in a typical time range of Xray Free-electron laser pulses. The results shows the fast dissipation of hydrogen atoms, bond shifting within the molecules during certain ionization degrees and the dissociation of the protein backbone after 20 fs.

Les travaux décrits dans ce manuscrit concernent la synthèse, l’étude structurale et l’évaluation biologique d’oligopeptides abiotiques incorporant le gamma-aminoacide hétérocyclique nommé ATC (acide-4-Aminométhyl-1,3-Thiazole-5-Carboxylique). Les ATCs sont construits autour d’un noyau thiazole et présentent deux points de diversité structurale. De précédents travaux ont déterminé que la présence du noyau thiazole entre les positions alpha et béta bloquait l'angle zéta autour de 0°, structurant les homo-oligomères de poly-(S)-ATCs en une hélice 9 droite et les faisant ainsi entrer dans le domaine des foldamères. Dans une première partie, nous avons entrepris de développer une voie de synthèse simple, flexible et énantiosélective permettant d’obtenir les ATCs stéréochimiquement purs sur une échelle de plusieurs grammes à partir d'alpha-aminoacides commerciaux. L’introduction de la diversité chimique est réalisée via deux étapes-clés que sont la condensation croisée de Claisen et la réaction de cyclisation de Hantzsch. Puis l’identification des marqueurs de structuration RMN et IR-TF des oligomères d'ATCs a été mise à profit pour caractériser le repliement d’hétéro-oligomères combinant ATCs et alpha-aminoacides. Ainsi, une étude structurale par RMN, IR-TF, cristallographie RX et dichroïsme circulaire a démontré que l’enchaînement 1:1 (L)-alpha:(S)-ATC se structurait en un ruban, stabilisé par un réseau intramoléculaire de liaisons hydrogène bifides formant des pseudocycles à 9 et 12 chaînons. La distribution des chaînes latérales le long du squelette principal présente une forte analogie avec l’hélice alpha, ce qui pourrait constituer un atout majeur pour le développement de composés à finalité thérapeutique. La dernière partie de ce travail a porté sur la conception de pseudo-peptides amphipatiques pour des applications en temps qu'antimicrobiens. / This manuscript describes the synthesis, the structural study and the biological evaluation of abiotic oligopeptides incorporating the heterocyclic gamma-amino acid ATC (4-Aminomethyl-1,3-Thiazole-5 Carboxylic acid). This original block is built around a thiazole ring and displays two lateral chains. Previous work in our laboratory highlighted that the presence of the thiazole ring between the positions alpha and beta implied that zeta angle was blocked around 0°, thus structuring poly-(S)-ATCs homo-oligomers in a right-handed 9-helix foldamer. First, development of a simple, flexible and enantioselective synthesis on a few grams scale has allowed to get access of a highly diverse ATC library from commercial alpha-amino acids. Introduction of the chemical diversity occurs via two key steps implying a cross-Claisen condensation and a Hantzsch cyclization. Then identification of NMR and FT-IR structural markers of ATC-containing oligomers was used to characterize the folding propensity of hybrid α:ATC oligomers. We demonstrated that 1:1 (L)-alpha:(S)-ATC heterochiral oligomers are structured in solution in a new ribbon-like shape stabilized by a bidentate intramolecular hydrogen bond network forming 9- and 12-membered pseudorings. The distribution of lateral chains along the main skeleton shows a high analogy with alpha-helix thus constituting a major advantage for potential medicinal applications. The last part of this work has focused on the design of amphipathic ATC-containing pseudo-peptides as antimicrobial agents.

The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

By applying the principles of Darwinian natural selection in the laboratory, directed evolution has become a powerful practical approach to study enzymes and optimize them to catalyze industrially relevant transformations. In this thesis, I applied this strategy to the engineering of amino acid dehydrogenases for biocatalysis of chiral amines, focusing on two crucial features for successful directed evolution experiments. A first key aspect is the development of technologies allowing the screening of large libraries of enzyme variants to explore sequence space efficiently. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsions has recently led to the development of ultrahigh-throughput screening platforms that allow sorting of more than 106 variants per hour. So far, these microfluidic droplet sorters have relied exclusively on fluorescent readouts. To further extend the range of applications toward enzymes for which no fluorescent assays are available, I successfully developed a sorting module based on absorbance detection. Using this new module, microdroplets could be sorted based on an absorbance readout at rates of up to 1 million droplets per hour. To demonstrate the utility of this module for protein engineering, three rounds of directed evolution were performed to improve a poorly stable NAD+ dependent phenylalanine dehydrogenase (PheDH) toward its native substrate. Five hits showed increased activity (improved up to 10-fold in lysate; kcat increased >3.5-fold), soluble protein expression levels (>2.5-fold) and thermostability (Tm, 8 °C higher). To increase the sensitivity of the device (3–4 orders of magnitude lower than fluorescence assays) for detection of enzymes with limited stability and low turnovers, an extra step of growth in droplets from single cell encapsulation, followed by piconinection of substrates and lysis agents was implemented. As a result, a fivefold signal enhancement over background was achieved, for an amine dehydrogenase (AmDH) reaction shown to be undetectable in a droplet single cell assay. Second, I investigated how mutational robustness may correlate with protein stability and lead to successful hits after mutagenesis and screening. To examine this issue, I initially investigated various approaches (including ancestral resurrection and computational design) to identify stabilized PheDH variants. One such variant (dubbed Pross 4) showed increased expression levels (>3.3-fold) and thermostability (Tm, 13 °C higher) compared to the wild-type PheDH. I further compared the mutational tolerance and the hit rate between PheDH and Pross 4 by generating variant libraries focused on key active site residues and screening them for improved AmDH activity. The Pross 4 background generated 6.4 times more active variants than the PheDH background, the best hits displaying increased activity (up to 2.5-fold in lysate; kcat/KM increased up to 8-fold) compared to previously engineered AmDHs with the PheDH scaffold. In conclusion, this work highlights how directed evolution experiments could be designed for increased success rates, by combining reliable high-throughput screens with careful choice of evolutionary robust starting points.

Resumo: O presente estudo teve por objetivo avaliar o efeito do uso de diferentes níveis de lisina e de metionina + cistina digestíveis durante o período pós - muda, sobre a recuperação corporal, desempenho, qualidade de ovos e morfometria do aparelho reprodutor, fígado e pâncreas de poedeiras comerciais no segundo ciclo de produção. Foram utilizadas 432 poedeiras comerciais da linhagem Isa Brown, com 72 semanas de idade, distribuídas em 54 parcelas, em um delineamento inteiramente casualizado com seis tratamentos e nove repetições de oito aves cada. Durante o descanso foram utilizados seis rações cujos níveis de lisina e metionina + cistina digestíveis variaram: 0,48% de lisina digestível e 0,43% de metionina+cistina digestíveis; 0,48% de lisina digestível e 0,47% de metionina+cistina digestíveis; 0,48% de lisina digestível e 0,52% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,50% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,56% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,62% de metionina+cistina digestíveis.Os dados obtidos foram submetidos à análise de variância e em caso de efeito significativo, a comparação de médias foi realizada a 5% de probabilidade através do teste de Tukey. Os diferentes níveis de lisina e de metionina+cistina digestíveis das dietas de descanso, determinaram efeitos significativos sobre os parâmetros de desempenho das aves. Observou-se que, o nível de 0,56% de lisina e 0,56% de metionina + cistina digestíveis, proporcionou maior peso dos ovos durante o segundo ciclo de produção / Abstract: This study was conducted to evaluate the effect of using different levels of lysine and methionine + cystine, about the body recovery, performance and egg quality of laying hens in the post molt. It was used four hundred and thirty two hens of Isa Brown strain, with 72 weeks of age, distributed in 54 cages in a completely randomized design with six treatments and nine replicates of eight birds each. During the rest period, were used six diets with different levels of digestible lysine and methionine + cystine. The values ranged from: 0.48% digestible lysine and 0,43% methionine + cystine; 0.48% digestible lysine and 0.47% methionine + cystine; 0.48% digestible lysine and 0.52% methionine + cystine; 0.56% digestible lysine and 0.50% methionine + cystine; 0.56% digestible lysine and 0, 56% methionine + cystine; 0.56% digestible lysine and 0.62% methionine + cystine. The data were subjected to analysis of variance and in case of significant effect, the comparison of means was performed at 5% probability by Tukey test. The different levels of lysine and methionine + cystine diets of rest have determined significant effects on the performance parameters of laying hens. It was observed that the level of 0.56% lysine and 0.56% methionine + cystine, resulted in greater weight of eggs during the second production cycle / Orientador: Otto Mack Junqueira / Coorientador: Silvana Martinez Baraldi Artoni / Banca: Antônio Carlos de Laurentiz / Banca: Rosimeire da Silva Filardi / Mestre

SILVA, José Gláucio da. Estudo dos modos normais de vibração de cristais de DL-Leucina por meio de Espectroscopia Raman. 2011. 60 f. Dissertação (Mestrado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by francisco lima (admir@ufc.br) on 2014-06-27T18:47:56Z No. of bitstreams: 1 2011_dis_jgdasilva.pdf: 13773993 bytes, checksum: 6087d8c6d1d8b72e3c3e0fb23080c962 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2014-08-06T17:58:17Z (GMT) No. of bitstreams: 1 2011_dis_jgdasilva.pdf: 13773993 bytes, checksum: 6087d8c6d1d8b72e3c3e0fb23080c962 (MD5) / Made available in DSpace on 2014-08-06T17:58:17Z (GMT). No. of bitstreams: 1 2011_dis_jgdasilva.pdf: 13773993 bytes, checksum: 6087d8c6d1d8b72e3c3e0fb23080c962 (MD5) Previous issue date: 2011 / Measures of Raman scattering were made on DL-leucine crystals at room temperature aiming to classify the normal vibration modes, based on group theory studies, as well as the classification of normal modes of other amino acid crystals. A study on the behavior of the DL-leucine normal modes with the temperature between 16 and 296K shows that there aren’t considerable changes on the Raman specters. This means the crystal structure is stable at the considered temperature interval, similarly to what was observed on other amino acid crystals such as L-alanine, L-isoleucine and glycine; however this stability is different than what was observed on the L-leucine crystal, which shows structural change at low temperature. / Foram realizadas medidas de espalhamento Raman em cristais de de DL-leucina à temperatura ambiente com o objetivo de classificar os modos normais de vibração, baseado no estudo de teoria de grupo, bem como a classificação dos modos normais de outros cristais de aminoácidos. Um estudo do comportamento dos modos normais da DL-leucina com a temperatura, entre 16 e 296 K, mostra que não ocorrem grandes mudanças nos espectros Raman. Isto significa que a estrutura do cristal é estável no intervalo de temperatura considerado, em analogia com que foi observado em outros cristais de aminoácidos como a L-alanina, a L-isoleucina, e a glicina; entretanto esta estabilidade é diferente no que foi observado no cristal de L-leucina, que apresenta mudança estrutural a baixa temperatura.

GUSMÃO, Gustavo Oliveira de Meira. Estudo das propriedades vibracionais do aminoácido DL-metionina por Espectroscopia Raman. 2014. 87 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2014-10-31T21:05:15Z No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2014-10-31T21:06:54Z (GMT) No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) / Made available in DSpace on 2014-10-31T21:06:54Z (GMT). No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) Previous issue date: 2014 / In this work, DL-methionine crystals (C5H11NO2S) are investigated by Raman spectroscopy varying the thermodynamic parameters temperature and pressure. Raman scattering measurements were performed on DL-methionine amino acid (C5H11NO2S) in the form at room temperature in the spectral region between 40 and 3200 cm-1. Subsequently the vibrational modes and wave numbers of the isolated methionine molecule through density functional theory (DFT) calculations were computed by implementing the exchange-correlation functional B3LYP and the basis set of 6-31 G (d, p) with the aid the Gaussian 03. The computational calculations reproduced the characteristics of the material in good agreement with the experimental spectrum. Based on this agreement, it was possible to associate the observed wave numbers with atomic displacements in the molecules. Also for the molecule of DL-methionine calculations distribution of potential energy PED were performed, allowing classifying eigenmodes with greater precision and confront those that have been reported in the literature. Measurements of Raman scattering in the crystal of DL-methionine in the spectral region between 50 cm-1 and 3200 cm-1 were performed from room temperature to the temperature of 10 K. In the spectra, no significant changes were observed, only temperature effects associated with decreasing anharmonicity. In the Raman spectroscopy experiments varying the temperature between 298 K and 443 K, it was found that the crystal of DL-methionine has undergone a structural phase transition at around 338 K, which were detected in the Raman spectra through the changes exhibited by the peaks associated with vibrations mainly attributed to carboxyl (CO2-), NH3+ group, CS and CC bonds and CH2 and CH3 groups. The analysis of the Raman spectra obtained after returning to room temperature revealed the crystal DL-methionine retrieves the beta phase. The experiment of thermal analysis confirms the structural phase transition suggested by Raman spectroscopy experiments at high temperatures. Raman scattering as a function of hydrostatic pressure measurements were done in the DL-methionine crystal. The experiments were performed in the spectral range between 50 cm-1 and 1200 cm-1 compressing the sample from atmospheric pressure up to the pressure of 5.1 GPa and then decompressing it to atmospheric pressure. Changes related to the vibrations of the CO2- and NH3+ units and external modes changes show that the crystal undergoes a structural phase transition at 1.5 GPa involving some of the hydrogen bonds. The results of the decompression show that the phase transition is reversible. / Neste trabalho, cristais de DL-metionina (C5H11NO2S) são investigados através de espectroscopia vibracional variando-se os parâmetros termodinâmicos temperatura e pressão. Medidas de espalhamento Raman foram realizadas no aminoácido DL-metionina (C5H11NO2S) na forma à temperatura ambiente na região espectral entre 40 e 3200 cm-1. Posteriormente foram computados os modos vibracionais e os números de onda da molécula isolada da DL-metionina na forma (C5H11NO2S) através de cálculos de teoria do funcional de densidade (DFT), implementando o funcional de troca e correlação B3LYP e a função de base 6-31 G(d,p) com o auxílio do programa Gaussian 03. Os cálculos computacionais reproduziram as características do material em boa concordância com o espectro experimental. Com base neste acordo, foi possível associar os números de onda observados aos deslocamentos atômicos nas moléculas. Ainda para a molécula de DL-metionina foram realizados cálculos de distribuição de energia potencial PED, o que possibilitou classificar os modos normais de vibração com maior precisão e confrontar com as que foram reportadas na literatura. Foram realizadas medidas de espalhamento Raman no cristal de DL-metionina na região espectral entre 50 cm-1 e 3200 cm-1 desde a temperatura ambiente até a temperatura de 10 K. Nos espectros não foram observadas mudanças significativas, apenas redução dos efeitos de anarmonicidade que se refletiu na mudança de intensidades e larguras de linha. Nos experimentos de espectroscopia Raman variando a temperatura entre 298 K e 443 K, verificou-se que o cristal de DL-metionina sofreu uma transição de fase estrutural em torno de 338 K, as quais foram detectadas no espectro Raman através das mudanças nos picos associados principalmente às vibrações atribuídas ao grupo carboxila (CO2-), e ao grupo anino NH3+, e à ligações CS e CSC e grupos CH2 e CH3. A análise dos espectros Raman obtidos após o retorno a temperatura ambiente revelaram que o cristal de DL-metionina recupera a fase inicial. O experimento de análise térmica confirmou a transição de fase estrutural sugerida pelos experimentos de espectroscopia Raman a altas temperaturas. Medidas de espalhamento Raman em função da pressão hidrostática foram realizadas no cristal de DL-metionina. Os experimentos foram feitos no intervalo espectral entre 50 cm-1 e 1200 cm-1 comprimindo a amostra desde a pressão atmosférica até a pressão de 5,1 GPa e em seguida descomprimindo-a até a pressão atmosférica através do uso de uma célula de pressão a extremos de diamante. Mudanças observadas nos modos externos e em modos relacionados às vibrações das unidades CO2 e NH3+ evidenciaram que o cristal sofre uma transição de fase estrutural em 1,5 GPa, envolvendo ligações de hidrogênio. Os resultados obtidos na descompressão da amostra mostraram que a transição de fase é reversível.

GUSMÃO, Gustavo Oliveira de Meira. Estudo das propriedades vibracionais do aminoácido DL-metionina por espectroscopia Raman. 2014. 87 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-10-15T21:42:10Z No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-10-20T20:53:15Z (GMT) No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) / Made available in DSpace on 2015-10-20T20:53:15Z (GMT). No. of bitstreams: 1 2014_tese_gomgusmao.pdf: 3607583 bytes, checksum: 0a291595482cfd3c0bfa53179df9d6cf (MD5) Previous issue date: 2014 / In this work, DL-methionine crystals (C5H11NO2S) are investigated by Raman spectroscopy varying the thermodynamic parameters temperature and pressure. Raman scattering measurements were performed on DL-methionine amino acid (C5H11NO2S) in the form at room temperature in the spectral region between 40 and 3200 cm-1. Subsequently the vibrational modes and wave numbers of the isolated methionine molecule through density functional theory (DFT) calculations were computed by implementing the exchange-correlation functional B3LYP and the basis set of 6-31 G (d, p) with the aid the Gaussian 03. The computational calculations reproduced the characteristics of the material in good agreement with the experimental spectrum. Based on this agreement, it was possible to associate the observed wave numbers with atomic displacements in the molecules. Also for the molecule of DL-methionine calculations distribution of potential energy PED were performed, allowing classifying eigenmodes with greater precision and confront those that have been reported in the literature. Measurements of Raman scattering in the crystal of DL-methionine in the spectral region between 50 cm-1 and 3200 cm-1 were performed from room temperature to the temperature of 10 K. In the spectra, no significant changes were observed, only temperature effects associated with decreasing anharmonicity. In the Raman spectroscopy experiments varying the temperature between 298 K and 443 K, it was found that the crystal of DL-methionine has undergone a structural phase transition at around 338 K, which were detected in the Raman spectra through the changes exhibited by the peaks associated with vibrations mainly attributed to carboxyl (CO2-), NH3+ group, CS and CC bonds and CH2 and CH3 groups. The analysis of the Raman spectra obtained after returning to room temperature revealed the crystal DL-methionine retrieves the beta phase. The experiment of thermal analysis confirms the structural phase transition suggested by Raman spectroscopy experiments at high temperatures. Raman scattering as a function of hydrostatic pressure measurements were done in the DL-methionine crystal. The experiments were performed in the spectral range between 50 cm-1 and 1200 cm-1 compressing the sample from atmospheric pressure up to the pressure of 5.1 GPa and then decompressing it to atmospheric pressure. Changes related to the vibrations of the CO2- and NH3+ units and external modes changes show that the crystal undergoes a structural phase transition at 1.5 GPa involving some of the hydrogen bonds. The results of the decompression show that the phase transition is reversible. / Neste trabalho, cristais de DL-metionina (C5H11NO2S) são investigados através de espectroscopia vibracional variando-se os parâmetros termodinâmicos temperatura e pressão. Medidas de espalhamento Raman foram realizadas no aminoácido DL-metionina (C5H11NO2S) na forma à temperatura ambiente na região espectral entre 40 e 3200 cm-1. Posteriormente foram computados os modos vibracionais e os números de onda da molécula isolada da DL-metionina na forma (C5H11NO2S) através de cálculos de teoria do funcional de densidade (DFT), implementando o funcional de troca e correlação B3LYP e a função de base 6-31 G(d,p) com o auxílio do programa Gaussian 03. Os cálculos computacionais reproduziram as características do material em boa concordância com o espectro experimental. Com base neste acordo, foi possível associar os números de onda observados aos deslocamentos atômicos nas moléculas. Ainda para a molécula de DL-metionina foram realizados cálculos de distribuição de energia potencial PED, o que possibilitou classificar os modos normais de vibração com maior precisão e confrontar com as que foram reportadas na literatura. Foram realizadas medidas de espalhamento Raman no cristal de DL-metionina na região espectral entre 50 cm-1 e 3200 cm-1 desde a temperatura ambiente até a temperatura de 10 K. Nos espectros não foram observadas mudanças significativas, apenas redução dos efeitos de anarmonicidade que se refletiu na mudança de intensidades e larguras de linha. Nos experimentos de espectroscopia Raman variando a temperatura entre 298 K e 443 K, verificou-se que o cristal de DL-metionina sofreu uma transição de fase estrutural em torno de 338 K, as quais foram detectadas no espectro Raman através das mudanças nos picos associados principalmente às vibrações atribuídas ao grupo carboxila (CO2-), e ao grupo anino NH3+, e à ligações CS e CSC e grupos CH2 e CH3. A análise dos espectros Raman obtidos após o retorno a temperatura ambiente revelaram que o cristal de DL-metionina recupera a fase inicial. O experimento de análise térmica confirmou a transição de fase estrutural sugerida pelos experimentos de espectroscopia Raman a altas temperaturas. Medidas de espalhamento Raman em função da pressão hidrostática foram realizadas no cristal de DL-metionina. Os experimentos foram feitos no intervalo espectral entre 50 cm-1 e 1200 cm-1 comprimindo a amostra desde a pressão atmosférica até a pressão de 5,1 GPa e em seguida descomprimindo-a até a pressão atmosférica através do uso de uma célula de pressão a extremos de diamante. Mudanças observadas nos modos externos e em modos relacionados às vibrações das unidades CO2 e NH3+ evidenciaram que o cristal sofre uma transição de fase estrutural em 1,5 GPa, envolvendo ligações de hidrogênio. Os resultados obtidos na descompressão da amostra mostraram que a transição de fase é reversível.

Dans la communauté scientifique, la glycosylation suscite un vif intérêt tant les relations existantes entre les sucres et les protéines sont étroites et sont impliquées dans de nombreux processus biologiques. De nombreuses méthodologies de synthèse ont été développées pour permettre la production de glycoprotéines sous forme homogènes nécessaires pour leur étude. Les formes S-glycosidiques sont d’un intérêt particulier grâce notamment à la réactivité de l’atome de soufre qui permet le couplage spécifique sucre – acide aminé et qui confère à la liaison une relative résistance à l’hydrolyse acido/basique et enzymatique. Dans le cadre de ce projet nous avons souhaité utiliser des outils biocatalytiques pour proposer une nouvelle voie d’accès aux protéines S-glycosylées en s’appuyant sur le concept des thioglycoligases. Pour répondre à ce challenge, nous nous sommes intéressés à la production d’une banque de glycosidases natives à partir du génome de la bactérie thermophile Dictyoglomus thermophilum. Trois protéines originales ont pu être produites, caractérisées et criblées en activité transglycosylase. Parmi ces dernières la β-glycosidase DtGly s’est révélée être un biocatalyseur efficace en favorisant la synthèse d’une variété de glycosides d’alkyle et de glycosyl glycérol. Par la suite, neuf mutants thioglycoligases ont été produits par mutagénèse dirigée à partir des glycosidases natives. A l’issue de leur criblage en activité thioligase, le mutant de DtGly E159Q a été retenu pour la synthèse de S-glycoconjugués. Une étude de relation structure/activité menée sur l’enzyme mutée nous a conduit à la synthèse d’un acide aminé non-naturel, la Thiotyrosine. L’essai de glycosylation de l’analogue estérifié de cette Thiotyrosine par DtGly E159Q a été suivi par LC-MS et a permis de mettre en évidence la formation du produit de thioglucosylation. Ce premier résultat préliminaire s’avère très prometteur pour la validation du concept de S-glycosylation d’un acide aminé catalysée par une thioglycoligase. / Glycosylation, one of the most complex co- and post-translationnal modifications of proteins, is of utmost importance for the scientific community as carbohydrates and proteins are closely related and involved in numerous diseases. The need to access homogeneous glycoproteins allows the development of a wide range of synthetic methods. Among them S-glycosidic forms have been attractive thanks to the reactivity of sulfur atom which allows the specific sugar – amino acid conjugation and confers a relative resistance through acido/basic or enzymatic hydrolysis. In this project we attempt to take advantage of natural tools to develop an enzymatic methodology to prepare S-glycosylated proteins through the thioglycoligase concept. In order to take up this challenge we first generate an enzymatic pool of wild type glycosidases from the thermophilic bacteria Dictyoglomus thermophilum. Activity screening of the three proteins produced and characterized allows the isolation of the β-glycosidase DtGly with good transglycosylation activity, thus affording a variety of alkyl glycosides and glyceroglycosides. Next we generate nine thioglycoligase mutants through site-directed mutagenesis and after screening of activity, the variant DtGly E159Q was selected to produce S-glycoconjugates. Finally, DtGly E159Q relation structure/activity studies led us to synthesize the unnatural amino acid Thiotyrosine. Enzymatic thioglucosylation assay catalyzed by DtGly E159Q was monitored by LC-MS and gave converging evidence of the successful formation of the S-glucosylated Thiotyrosine. This tremendous preliminary result is promising for further investigations of enzymatic S-glycosylation of amino acid using thioglycoligases as toolbox.

Submitted by RAFAEL MASSAMI SUZUKI null (rafael-msuzuki@hotmail.com) on 2016-04-25T16:27:49Z No. of bitstreams: 1 Dissertação Mestrado Rafael - Definitiva.pdf: 1828628 bytes, checksum: 7ab33dfa0c92841eda9e9c68bfc23a60 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-27T18:43:50Z (GMT) No. of bitstreams: 1 suziki_rm_me_jabo.pdf: 1828628 bytes, checksum: 7ab33dfa0c92841eda9e9c68bfc23a60 (MD5) / Made available in DSpace on 2016-04-27T18:43:50Z (GMT). No. of bitstreams: 1 suziki_rm_me_jabo.pdf: 1828628 bytes, checksum: 7ab33dfa0c92841eda9e9c68bfc23a60 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos relacionados a utilização de um determinado nutriente são importantes para se obter conhecimentos a respeito do aproveitamento deste nutriente pelo animal, uma vez que há nem tudo o que é fornecido, é aproveitado pelo mesmo. Dessa forma, o que foi de fato utilizado para o animal pode ser convertido em produção animal, como um acréscimo proteico nos frangos de corte, ou então a utilização de nutrientes para a produção de ovos em poedeiras. A utilização de aminoácidos marcados nos permite avaliar processos metabólicos em diferentes estudos nutricionais, tanto no processo de incorporação refletido no processo de turnover, quanto na deposição do aminoácido nos diferentes tecidos. Deste modo, o trabalho teve como enfoque a utilização da L-(15N) treonina que foi explorada por meio de três abordagens diferentes: verificar a distribuição deste aminoácido marcado em diferentes tecidos de frangos de corte por meio de um balanço de massas (Capítulo 2); calcular a eficiência de utilização nestes tecidos, ou seja, calcular qual foi o aproveitamento da L-(15N) treonina ingerida que foi depositada/incorporada a esses tecidos (Capítulo 2); e por fim, analisar o turnover da L-(15N) treonina para se analisar a incorporação do aminoácido no tecido (Capítulo 3). / Studies related to the utilization of a particular nutrient are important to obtain knowledge regarding the utilization of this nutrient by the animal, because only a part of the nutrient will be used. Thus, what was in fact used for the animal can be converted to livestock as an increase in protein broilers, or the use of nutrients to egg production of laying hens. The use of labeled amino acids allows us to evaluate metabolic processes in different nutritional studies, both in the incorporation process reflected in the turnover process, as the amino acid deposition in different tissues. Thus, the aim of the work was to focus the utilization of L-(15N) threonine, which was explored by three different approaches: verify the distribution of labelled amino acid in different tissues of broiler chickens through a mass balance (Chapter 2 ); calculate the utilization efficiency of these tissues or the L-(15N) threonine intake which has been deposited / incorporated into these tissues (Chapter 2); and finally, to analyze the turnover of L-(15N) for threonine analyzing the amino acid incorporation into the tissue (Chapter 3). / FAPESP: 2013/25761-4