Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.

Made available in DSpace on 2016-06-02T19:56:52Z (GMT). No. of bitstreams: 1 5338.pdf: 2812584 bytes, checksum: c4ea2f65f591bec0e6ad7105c6ec591e (MD5) Previous issue date: 2013-06-20 / Universidade Federal de Minas Gerais / In this work, it was studied the simultaneous isomerization and fermentation of xylose to ethanol (SIF) using xylose isomerase (XI) and S. cerevisiae co-immobilized in calcium alginate gel. XI was immobilized on chitosan gel activated with glutaraldehyde (IXI-Ch). The influence of the concentration of enzyme/yeast in the reactor, the pH, temperature and yeast strain on yield and selectivity in ethanol was studied. The concentrations of enzyme and yeast in the reactor were varied by changing the mass of IXI-Ch and yeast per gram of alginate solution, maintaining fixed the ratios of biocatalyst weight: volume of medium in the reactor (1:1). The SIFs were carried out in batch with xylose (65g.L-1), antibiotics and other salts. The first experiment, with 16% Itaiquara® yeast and 5% enzyme biocatalyst (% wenzyme or yeast/wbiocatalyst) showed that pH drop occurred during the test, preventing full conversion of xylose, due to reduced enzyme activity. calcium carbonate (0.5-1.0%) was then included in the biocatalyst, which maintained the pH between 5,2 to 5,6, allowing complete conversion of the sugar at all concentrations tested (%Yeast -Enzyme in biocatalyst): 5-20, 17-5, and 10 yeast (Itaiquara ®) with 5, 10 and 20-. The maximum ethanol productivity, 2,44 ± 0,26g.L-1.h- 1 was obtained for the highest cell concentration and the highest selectivity ethanol/xylitol, 2,57 ± 0.4 and 2,42 ± 0,01 for the highest enzyme concentrations (10 and 20% with 10% yeast). These results indicated that the highest concentration of xylulose favored more selectivity to ethanol. Fermentation was then performed using no enzyme in biocatalyst with a prior isomerized syrup concentrated in xylulose containing 58g.L-1 xylulose and 9g.L-1 xylose and another with xylose only. At first, xylulose was completely assimilated in 5 hours, xylose was barely consumed in both assays, and ethanol selectivity was lower than that obtained in the SIF tests. Xylitol show thus to be produced mainly from xylulose and selectivity contrary to expectations did not directly increase with increasing xylulose concentration, indicating that the formation of ethanol/xylitol depends not only on external xylulose, and it is probably finely regulated in yeast. The concentrations of enzyme and yeast 20 and 10% (equivalent to 100gderived.L-1 reactor and 50gwd.L-1 reactor) were selected as the best, which were used to study the influence of pH and temperature, and also different strains. The increase of initial pH from 5.6 to 6.5 did not improve the productivity, yield, neither selectivity in ethanol. Temperatures tested for Itaiquara ® were 32, 35 and 37 ° C, and for industrial strains CAT-1 and BG-1: 32, 37 and 40 ° C. Viability remained above 90% for all assays at 24 hours. All three strains showed increased selectivity in ethanol with temperature reduction, obtaining the maximum selectivity for industrial strains (3,06 ± 0,24 - CAT-1 and 3,19 ± 0,11 BG-1) with yield and productivity equal or greater than those obtained in higher temperatures. At 32 ° C and pH 5.6, Itaiquara ® showed lower conversion time, but lower selectivity, while the BG-1, demonstrated the highest selectivity, but low conversion and productivity. The strain CAT-1 combines high productivity, 2,17 ± 0,17 g.L-1.h-1, and selectivity, 3,06 ± 0,24, with 90% conversion in 9 hours, 32 ° C, which is apparently the best performance among the tested yeasts. The results were very promising, indicating the technical feasibility of producing ethanol from xylose with the biocatalyst developed. / Neste trabalho foi estudada a simultânea isomerização e fermentação de xilose a etanol (SIF) usando xilose isomerase (XI) e S. cerevisiae coimobilizadas em gel de alginato de cálcio. XI foi imobilizada em gel de quitosana ativado com glutaraldeido (IXI-Ch).. Foram estudadas as influências das concentrações de enzima/levedura no reator, do pH, da temperatura e da linhagem de levedura na produtividade e na seletividade em etanol. As concentrações de enzima e levedura no reator foram variadas mudando-se a massa de IXI-Ch e levedura por grama de solução de alginato, mantendo-se fixas as proporções 1:1 massa de biocatalisador:volume de meio no reator. As SIFs foram realizadas em batelada com xilose (~65g.L-1), antibiótico e outros sais. O primeiro experimento realizado, biocatalisador com 16% levedura Itaiquara® e 5% enzima (% menzima ou levedura/mbiocatalisador), mostrou que ocorria queda de pH durante o ensaio, impedindo conversão total da xilose, devido à redução da atividade enzimática. Foi incluído carbonato de cálcio 0,5-1,0% no biocatalisador, o que manteve o pH entre 5,2-5,6, permitindo total conversão do açúcar, em todas as concentrações testadas (%Levedura-Enzima no biocatalisador): 5-20, 17-5 e 10 levedura (Itaiquara®)com 5, 10 e 20% enzima. A máxima produtividade em etanol, 2,44 ± 0,26g.L-1.h-1, foi obtida para a mais alta concentração celular e a mais alta seletividade etanol/xilitol, 2,57± 0,4 e 2,42± 0,01, para as mais altas concentrações de enzima (10 e 20% com 10% levedura). Esses resultados indicavam que quanto mais alta a concentração de xilulose, mais favorecida a seletividade em etanol. Foi então realizada uma fermentação usando o biocatalisador sem enzima, com um xarope previamente isomerizado e concentrado em xilulose contendo 58g/L de xilulose e 9g/L xilose e outro apenas com xilose. No primeiro, xilulose foi totalmente assimilada em 5 horas, xilose foi pouco consumida nos dois ensaios, e a seletividade em etanol foi menor que a obtida nos ensaios SIF. Xilitol mostrou, assim, ser produzido majoritariamente a partir de xilulose e contrariamente ao esperado a seletividade não aumenta diretamente com o aumento da concentração de xilulose, indicando que o metabolismo etanol/xilitol não depende apenas da concentração externa de xilulose, devendo ser finamente regulado dentro da levedura. Selecionaram-se as concentrações de enzima e levedura de 20 e 10% (equivalente a 100gderivado.L-1 reator e 50gms.L-1 reator) como as melhores, as quais foram utilizadas para estudo da influência do pH e da temperatura e ainda de diferentes linhagens. O aumento do pH inicial do meio de 5,6 para 6,5 não favoreceu a produtividade, rendimento e nem a seletividade em etanol. As temperaturas testadas para Itaiquara® foram: 32, 35 e 37°C; e para as linhagens industriais CAT-1 e BG-1: 32, 37 e 40°C. A viabilidade manteve-se acima de 90% para todos os ensaios em 24 horas. As três linhagens mostraram aumento da seletividade em etanol com a redução da temperatura, obtendo-se a máxima seletividade para as linhagens industriais (3,06± 0,24- CAT-1 e 3,19± 0,11 BG-1), com rendimento e produtividade iguais ou maiores que os obtidos nas temperaturas maiores. A 32°C e pH 5,6, Itaiquara® apresentou menor tempo de conversão, mas a menor seletividade, já a BG-1, obteve maior seletividade, mas baixa conversão, rendimento e produtividade. A linhagem CAT-1 alia alta produtividade, 2,17 ± 0,17 (g.L-1.h-1), e seletividade, 3,06 ± 0,24, com 90% de conversão em 9 horas, 32°C, sendo aparentemente a de melhor desempenho dentre as testadas. Os resultados foram muito promissores, indicando viabilidade técnica de produção de etanol a partir de xilose com o biocatalisador desenvolvido.

Made available in DSpace on 2016-06-02T19:56:44Z (GMT). No. of bitstreams: 1 3706.pdf: 2821060 bytes, checksum: 5975ad59eeeedfbd6e16578292306171 (MD5) Previous issue date: 2011-08-01 / Universidade Federal de Sao Carlos / The objective of this study was to verify the influence of the Si/Al ratio (11.5, 15.0, 25.0, 40.0 and 140.0) in zeolite ZSM-5 in the conversion, activity, selectivity and stability as bifunctional catalysts (Pt/H-ZSM-5) applied to the isomerization reaction of n-hexane. To prepare the bifunctional catalysts, initially the zeolites were submitted, successively, to ion exchange with ammonium cations, ion exchange cations with platinum, calcination and activation process. From the results of the isomerization of n-hexane, it was observed that with the increase of the Si/Al ratio, that is, decreasing the amount of aluminum, there was a reduction in activity and conversion. This is due to decrease the number of acid sites present, responsible to isomerize the carbocation generated in these sites. In contrast, the increase of the Si/Al ratio leads to a better selectivity to the formation of isomers. Among the catalysts, the Pt/H-ZSM-5 (15.0) showed the best result of conversion and activity. In order to compare the Pt/H-ZSM-5 catalysts, the reaction was carried out using the isomerization catalyst Pt/H-Beta (9.0). This catalyst had the best result that all the others, this result may possibly be due to increased acidity of the material and its morphological characteristics, such as type and diameter of pores etc. and/or the crystallite size of zeolites used. / O objetivo deste estudo foi verificar a influência da razão Si/Al (11,5, 15,0, 25,0, 40,0 e 140,0) na zeólita ZSM-5 através da conversão, atividade, seletividade e estabilidade de catalisadores bifuncionais (Pt/H-ZSM-5) aplicados a reação de isomerização do n-hexano. Para preparar os catalisadores bifuncionais, inicialmente as zeólitas foram submetidas, sucessivamente, a troca iônica das zeólitas com cátions amônio, troca iônica com cátions platina, processos de calcinação e ativação. A partir dos resultados da isomerização do nhexano observou que com o aumento da razão Si/Al, isto é, diminuição da quantidade de alumínio, obteve-se uma redução na atividade e conversão. Tal fato se deve a diminuição do número de sítios ácidos presentes, sendo que os mesmos são responsáveis por isomerizar os carbocátions gerados nos sítios. Em contrapartida, o aumento da razão Si/Al acarreta em uma melhor seletividade a formação dos isômeros. Dentre os catalisadores Pt/H-ZSM-5, o catalisador Pt/H-ZSM-5 (15,0) foi o que apresentou melhor resultado de atividade e conversão. A fim de comparar os catalisadores Pt/H-ZSM-5, a reação de isomerização do nhexano foi realizada utilizando o catalisador Pt/H-Beta (9,0). O mesmo obteve melhor resultado que todos os outros, podendo tal resultado ser, possivelmente, devido a maior acidez do material e suas características morfológicas, como tipo e diâmetro de poros etc. e/ou pelo tamanho do cristalito das zeólitas utilizadas.

Il est connu que les 4,5-méthano-L-prolines ont des conformations et des caractéristiques différentes que la L-proline. L’aplatissement du cycle pyrrolidine dans la proline joue un rôle important dans sa stabilité et la conformation spatiale des analogues 4,5-méthano- de la L-proline. Ainsi, des études approfondies ont été menées pour analyser les différences de structure et les modèles de pliage des cis- et trans-4,5-méthano-L-prolines et leurs oligomères en corrélation avec la délocalisation électronique n→π∗ entre les carbonyles adjacents. Dans cette thèse, la stabilisation n→π∗ entre les groupes carbonyl d’une amide et un ester a été analysée, divulguant des propriétés structurelles supplémentaires des 4,5-méthano-L-prolines. Les études spectroscopiques en RMN ont révélé les caractéristiques spatiales des cis- and trans-4,5-méthano-L-prolines corrélant avec l’équilibre d’isomérisation de l’amide trans : cis dans des environnements différents. En outre, le mécanisme de la cyclisation intramoléculaire des dimères cis-cis, trans-trans, et du mélange trans,cis-4,5-méthano-L-proline dans la formation des dicétopipérazines a révélé l'importance de la stabilisation n→π∗ sur les caractéristiques conformationnelles et de pliage. Des études de modélisation DFT ont également été menées afin de compléter nos résultats et de fournir des explications soutenant nos résultats expérimentale. / It is well known that 4,5-methano-L-prolines have different conformational properties and characteristics than proline. The flattening of the pyrrolidine ring plays a role in the stability and conformation of L-proline methanologs. Thus, extensive studies have been conducted to analyze the structural differences and folding patterns of cis- and trans-4,5-methano-L-prolines and their oligomers in correlation with the n→π∗ electron delocalization between the adjacent carbonyls of amide and ester groups. In this thesis, the n→π∗ stabilization of each derivative is analyzed, disclosing additional structural properties of 4,5-methano-L-prolines. NMR spectroscopic studies revealed the spatial characteristics of cis- and trans-4,5-methano-L-prolines in correlation to the amide trans : cis isomerization equilibrium in different solvents. In addition, the tendency of diketopiperazine formation from the cis-cis, trans-trans, and mixed trans,cis-4,5-methano-L-proline dimers reflected the importance of the n→π∗ stabilization on the conformation and folding characteristics.

Among the peptidyl prolyl isomerases, the Cyclophilin family of proteins has been linked to various cellular activities such as regulation of homeostasis, mitochondrial permeability, and cell death. Their functionality spans throughout the cell and throughout all cell types as different isoforms. Previous studies done on Cyclophilin A revealed an interesting contact ensemble when bound to a substrate. Because of the similarity of CypA to its homologues, it is believed that they too will exhibit the same contact dynamics. We have defined the dynamics of cyclophilin isoforms through Molecular Dynamics simulations and determined their contact dynamics, characterizing their contact ensembles, and their relative dynamical conservation to each other.

La réaction d'aldolisation est une des méthodes les plus importantes et plus utilisées pour former des liaisons C-C. La réaction tandem d'isomérisation-aldolisation catalytique d'alcools allyliques permet d'effectuer cette réaction avec de nombreux avantages synthétiques et nous avons préparé par ce moyen des beta-hydroxyacylsilanes à partir d'alpha-hydroxyallylsilanes. Tout d'abord, nous avons cherché à mettre au point une version catalytique asymétrique de cette réaction tandem et nous avons aussi synthétisé des beta-hydroxyacylsilanes par aldolisation directe. Ensuite, nous avons utilisé des alpha-hydroxyallylsilanes pour préparer des aldolsalpha-silylés au moyen de réactions d'époxydation. Dans une troisième partie, nous avons synthétisé des aldols à partir de beta-hydroxyacylsilanes protégés de manière simple et efficace, ce qui nous a permis d'effectuer des réactions d'aldolisation itératives. Nous avons illustré le potentiel de cette méthode par la synthèse d'un fragment de (±)-pironetine. Enfin, nous avons synthétisé, à partir des mêmes beta-hydroxyacylsilanes protégés, des éthers d'énol silylés qui ont été ensuite utilisés avec succès comme substrats pour des réactions de Mukaiyama. / The aldol reactions is one of the most important and commonly used methods to form C-C bonds. The catalytic tandem isomerization-aldol reaction of allylic alcohols allows to perform this reaction with many synthetic advantages. Thus, we have prepared by this method beta-hydroxyacylsilanes from alpha-hydroxyallylsilanes. First, we have attempted to develop a catalytic asymmetric version of this reaction, and also to synthesize beta-hydroxyacylsilanes by direct aldol reaction. Then, we used alpha-hydroxyallylsilanes to prepare alpha-silyl aldols trough epoxidation reactions. Next, we synthesized aldols from protected beta-hydroxyacylsilanes in a simple and efficient fashion which allowed us to perform iterative aldol reactions. We have illustrated the potential of this method by the synthesis of a fragment of (±)-pironetine. Finally, we have synthesized, from the same protected beta-hydroxyacylsilanes, silyl enol ethers that next have been used successfully as substrates for Mukaiyama aldol reactions.

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