Red fluorescent proteins (RFPs) are used extensively in biological research because their longer emission wavelengths are less phototoxic and allow deeper imaging of animal tissue. However, far-red RFPs generally display low brightness, emphasizing the need to develop brighter variants. Here, we investigate three approaches to rigidify the RFP chromophore to increase the quantum yield, and thereby brightness. We first used computational protein design on a maturation-efficient mRojo-VHSV variant previously engineered in our lab to introduce a Superdecker motif, a parallel pi-stack comprising aromatic residue side chains and the phenolate moiety of the chromophore, which we hypothesized would enhance chromophore packing and reduce non-radiative decay. The best mutants identified showed up to 1.7-fold higher quantum yield at pH 9, relative to their parent protein. We next postulated that brightness could be further increased by rigidifying the chromophore via branched aliphatic residues. Computational protein design was performed on a dim mCherry variant, mRojoA, followed by directed evolution on the brightest mutant. The combination of these methodologies yielded mSandy2, the brightest Discosoma-derived monomeric RFP with an emission maximum above 600 nm. Finally, we aimed to increase brightness by focusing on positions where residue rigidity correlated to quantum yield in mCherry-related RFPs according to NMR data that had been previously acquired in our lab. Combinatorial site-saturation mutagenesis was performed on two different surface patches of mCherry at positions 144/145/198 and 194/196/220. Our results demonstrated that surface residues may not be adequate targets for this approach. Altogether, the work herein presents unique rational design methodologies that can be used to increase brightness in RFPs.

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京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18288号 / 工博第3880号 / 新制||工||1595(附属図書館) / 31146 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 一義, 教授 田中 庸裕, 教授 佐藤 啓文 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21315号 / エネ博第371号 / 新制||エネ||73(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー応用科学専攻 / (主査)教授 松田 一成, 教授 佐川 尚, 教授 大垣 英明 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

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No description available.

Foram sintetizados, purificados e caracterizados os peróxidos cíclicos: peróxido de difenoíla (1), dimetil-1,2-dioxetanona (2), e spiro-adamantil α-peróxi lactona (3), e determinados seus parâmetros de quimiluminescência. Foram redeterminados os rendimentos quânticos singlete na decomposição catalisada de 1 e 2 em condições análogas as utilizadas anteriormente na literatura, obtendo-se valores várias ordens de grandeza menores que os inicialmente relatados para estes sistemas padrão para a formulação do mecanismo Luminescência Iniciada Quimicamente por Troca de Elétron (Chemically Initiated Electron Exchange Luminescence - CIEEL). O efeito de gaiola de solvente, utilizando-se o sistema binário tolueno/difenilmetano, sobre o rendimento quântico na decomposição de 1 catalisada por rubreno, mostrou-se consideravelmente menor para este sistema intermolecular do que o observado no sistema intramolecular da decomposição induzida de 1,2-dioxetanos fenóxi-substituidos. Foram determinados pela primeira vez os parâmetros de quimiluminescência na decomposição unimolecular e catalisada da spiro-adamantil α-peróxi lactona (3), mostrando que esta é a α-peróxi lactona mais estável sintetizada e estudada até o momento. Este peróxido é sujeito à decomposição catalisada por ativadores adequados, envolvendo transferência de elétron, porém, com eficiência baixa devido a um impedimento estérico exercido pelo substituinte spiro-adamantila sobre a interação do peróxido com o ativador. O rendimento quântico singlete máximo obtido na decomposição catalisada de 3 é de 1%, valor consideravelmente menor que os determinados para sistemas intramoleculares. A decomposição dos peróxidos cíclicos 1 a 3 é catalisada por fenolatos como ativadores, porém, os rendimentos singlete obtidos nesta reação são baixos, entre 10-3 e 10-7 E mol-1. Os resultados obtidos com este sistema modelo intermolecular para a decomposição induzida por transferência intramolecular de elétron de 1,2-dioxetanos fenóxi-substituidos, demonstram claramente que esta última transformação quimiluminescente de alta eficiência deve ocorrer por uma retro-transferência de elétron intramolecular e não intermolecular como proposto na literatura, e como é necessariamente o caso no sistema modelo estudado no presente trabalho. / The cyclic peroxides diphenoyl peroxide (1), dimethyl-1,2-dioxetanone (2) and spiro-adamantyl α-peroxy lactone (3) were synthesized, purified and characterized, as well as its chemiluminescence parameters determined. The singlet quantum yields in the catalyzed decomposition of 1 and 2 were re-determined in experimental conditions equivalent to those formerly utilized in the literature, obtaining values various orders of magnitude lower than the ones initially reported for these model systems for the formulation of the Chemically Initiated Electron Exchange Luminescence\" (CIEEL) mechanism. The solvent-cage effect on the singlet quantum yields in the rubrene catalyzed decomposition of 1, using the binary system toluene / diphenylmethane, showed to be considerably lower for this intermolecular system than the one observed in the intramolecular system of the induced phenoxy-substituted 1,2-dioxetane decomposition. The chemiluminescence parameters in the unimolecular and catalyzed spiro-adamantyl α-peroxylactone (3) decomposition were determined for the first time, showing that 3 is the most stable α-peroxylactone synthesized and studied up to now. This peroxide is subject to catalyzed decomposition by appropriate activators involving electron transfer, however, with low efficiency due to the steric effect of the spiro-adamantyl substituents on the interaction of the peroxide with the activator. The maximum singlet quantum yield obtained in the catalyzed decomposition of 3 is 1 %, a value considerably lower than those determined for intramolecular systems. The decomposition of the cyclic peroxides 1 to 3 is catalyzed by fenolato ions as activators, thus the singlet quantum yields measured for this reaction are low, between 10-3 e 10-7 E mol-1. The results obtained for this intermolecular model system for the induced phenoxy-substituted 1,2-dioxetane decomposition demonstrate clearly that the latter highly efficient chemiluminescent transformation should occur by an intramolecular back-electron transfer and not by an intermolecular one as proposed in the literature, and as is necessarily the case in the model system studied in this work.

Les comètes présentent un grand intérêt à la fois pour la planétologie et pour l'exobiologie. En effet, ces corps primitifs du fait de leur petite taille et de leurs réservoirs éloignés du soleil, n'ont pas ou que très peu évolué depuis leur formation. L'étude des comètes peut donc permettre de mieux comprendre les processus physico-chimiques ayant eu lieu lors de la formation du Système Solaire. D'autre part, les analyses menées en 1986 dans l'environnement de la comète 1P/Halley ont montré l'existence, dans les grains cométaires d'une phase solide riche en composés organiques. Ainsi, les comètes ont vraisemblablement pu apporter sur la Terre primitive des composés organiques, et favoriser ainsi l'apparition de la Vie. Néanmoins la nature de cette matière organique reste encore très largement méconnue. Ces composés organiques ont vraisemblablement été formés à partir des glaces observées dans le milieu interstellaire et qui sont soumises à différentes sources d'énergie. Les objectifs du travail expérimental mené au cours de cette thèse ont donc été de caractériser les différentes étapes conduisant à la synthèse des composés organiques complexes contenus dans les comètes à partir des glaces interstellaires. En particulier, j'ai étudié i.) la quantification des glaces présentes autour des étoiles naissantes, ii.) les processus de photolyse auxquels elles sont soumises et iii.) la nature des composés organiques qui peuvent être produits durant ces processus. Les observations infrarouges ont permis de détecter de nombreuses molécules en phase condensée autour des étoiles naissantes. Afin de préciser l'abondance de ces molécules, j'ai mesuré les sections efficaces intégrées, aussi appelées forces de bandes, pour huit d'entre elles (H2O, CO, CO2, CH3OH, NH3, CH4, HCOOH and H2CO). En effet, ce paramètre spectroscopique est nécessaire à la quantification des molécules et certaines des valeurs présentes dans la littérature affichaient une grande dispersion. Les nouvelles mesures que j'ai effectuées, basées sur une revue bibliographique exhaustive des masses volumiques et des indices optiques dans le visible, confirment pour certaines molécules (CO2, CO, CH4, NH3) les valeurs utilisées pour déterminer leur abondance. Néanmoins, j'ai pu montrer que les abondances d'autres molécules dans les milieux astrophysiques restent encore très incertaines, en particulier pour CH3OH, H2CO et HCOOH. En phase condensée, la dépendance en longueur d'onde des processus de photolyse est encore très largement négligée. Grâce à l'utilisation de deux lampes VUV, dont l'irradiance spectrale a été préalablement caractérisée, j'ai pu mesurer, dans deux gammes de longueurs d'onde différentes, les rendements quantiques de production de C2H6 et de CO lors de la photolyse de CH4 et CO2. J'ai ainsi pu confirmer que les rendements quantiques dépendent bien de la longueur d'onde de photolyse. L'extrapolation des résultats expérimentaux acquis en laboratoire aux différents milieux astrophysiques nécessite donc une bonne connaissance des spectres VUV mis en jeu. L'objectif final des simulations expérimentales est de prédire la nature de la matière organique cométaire en reproduisant au mieux la chimie pouvant se dérouler dans les glaces interstellaires. Or, le méthane a été détecté en phase condensée dans le milieu interstellaire, mais son influence sur la chimie se déroulant dans des mélanges de glaces contenant les principales molécules interstellaires a été très peu étudiée. J'ai donc soumis un mélange H2O : CH3OH : NH3 : CH4 (10 : 1 : 1 : 2) à une photolyse de 26 heures puis à un chauffage. L'influence du méthane se manifeste par la présence de C2H6 après la photolyse à basse température. Lors du chauffage, le méthane et ses photoproduits semblent se sublimer. Avec ou sans CH4, la chimie à des températures supérieures à 200 K apparaît très similaire. J'en conclu donc que la présence de méthane ne modifie pas notablement la chimie des glaces lors des simulations / Comets are very interesting for planetology as well as for exobiology. On one hand, held in the furthest and coldest regions of our solar system and due to their small size, they might not have been altered since their formation. The study of comets should allow a better understanding of the physic-chemical processes occurring during the Solar system formation. On the other hand, the analysis performed in 1986 on the environment of 1P/Halley showed the presence, in the cometary dust, of organic matter. Thus, comets might have brought organics on primitive Earth which might have contributed to the apparition of life. Nevertheless, the nature of these organics is still not well-known. Cometary organics might have been synthesized from the ices detected in interstellar medium which are submitted to different energetic processes. The aims of the experimental work performed during this thesis are to characterize the different steps of the synthesis of complex organic matter contained in comets from the interstellar ices. I studied : i) The quantification of interstellar ices detected around young stellar objects ii) The characterization of the photolysis process to which ices are submitted and iii.) The nature of the organic compounds produced during these processes. Once mixtures and energetic processes are under control, we can make cometary organic analogs. Infrared observations have revealed the presence of several molecules in the solid phase around young stellar objects. To precise their molecular abundances, I have measured the integrated cross sections, also called band strengths, of 8 molecules (H2O, CO, CO2, CH3OH, NH3, CH4, HCOOH and H2CO). Indeed, this spectroscopic parameter is required for the quantification of these molecules and some values presented in literature are scattered. The new measurements performed during this thesis, which are based on a bibliographic review of densities and optical indices in the visible range, confirm the values already used for the quantification of CO2, CO, CH4, NH3. But this work also underlines that abundances of CH3OH, H2CO and HCOOH in interstellar medium are still uncertain. In the solid phase, wavelength dependence of photolysis is often neglected. Thanks to two VUV lamps, for which the spectral irradiances have been characterized, I measured the production quantum yield, in two wavelength ranges, of C2H6 and CO, during photolysis of CH4 and CO2 respectively. Thanks to this study, I point out that quantum yield depends on the photolysis wavelength. Thus, the extrapolation of the experimental results to different astrophysical medium implies a good knowledge of VUV spectra. The final objective of experimental simulations is to foresee the nature of cometary organic matter by reproducing, as realistic as possible, the chemistry occurring in interstellar ices. Methane has been detected in the solid phase in the interstellar medium, but few studies implying methane have been undertaken. Thus, I have photolyzed a mixture composed of H2O: CH3OH: NH3: CH4 (10:1:1:2) during 26 hours at low temperature and then I applied a heating process. The influence of initial methane in the ice chemistry is demonstrated by the presence of its main photoproducts, C2H6, after photolysis. But while increasing temperature, methane and its photoproducts seem to sublimate. Therefore, with or without methane, chemistry occurring at temperature higher than 200K seems to be very similar. I conclude than methane does not have significant influence on ice chemistry

Neste trabalho, estudou-se a degradação do bisfenol A (BPA) em solução aquosa através da fotólise direta sob radiação UV em 254 nm. Os experimentos foram realizados um reator fotoquímico tubular de vidro de 3,9 L, equipado com uma lâmpada de vapor de mercúrio de baixa pressão tubular concêntrica (36 W). Avaliaram-se os efeitos da taxa específica de emissão de fótons (0,87×10 18 ; 1,4×1018 e 3,6×1018 fótons L-1 s-1 ) e da concentração inicial de BPA (10 a 50 mg L-1 ), conforme um projeto experimental Doehlert. Os resultados dos experimentos indicaram que a degradação do BPA diminui com o aumento de sua concentração inicial, seguindo decaimento de pseudo primeira-ordem. A constante cinética de velocidade de degradação do BPA variou de 0,001 a 0,0066 min-1 , enquanto a porcentagem de degradação de BPA ao final de 120 minutos variou de 14 a 55%; já a remoção do carbono orgânico total variou entre 1,5 e 12,5%. Houve a formação de produtos persistentes e as espécies reativas de oxigênio (1 O2 oOH ) mostraram-se muito importantes durante a degradação do BPA. O rendimento quântico para fotólise de BPA foi de 0,0075 mol BPA mol fótons-1, determinado a partir dos dados experimentais através de um modelo matemático para degradação fotolítica do BPA em função do tempo. Para tanto, considerou-se um sistema de tratamento formado por um reator tubular e um tanque de mistura com recirculação entre eles, sendo o reator tubular tratado como uma série de três reatores contínuos de mistura perfeita (CSTR) associados entre si. Através da análise estatística dos dados experimentais para duas respostas (constante específica de degradação do BPA e porcentagem de degradação do BPA ao final de 120 minutos) avaliaram-se os efeitos das variáveis envolvidas (concentração inicial de BPA e taxa específica de emissão de fótons). / The aim of this study was to evaluate bisphenol A (BPA) degradation in aqueous solution by direct photolysis under UV radiation at 254 nm, considering the effects of the specific rate of photon emission (0.87×1018; 1.4×1018 and 3.6×1018 fótons L-1 s-1) and the initial BPA concentration (10-50 mg L-1), according to a Doehlert experimental design. The experiments were performed in a glass tubular photochemical reactor of 3.9 L equipped with a concentric low pressure mercury vapor lamp of 36 W. The experimental results indicated that BPA degradation decreases with increasing initial concentration, following pseudo first-order decay, and increases with increasing specific rate of photon emission. The values of the specific BPA degradation rate varied in the range 0.001-0.0066 min-1, while BPA percent degradation at 120 minutes of irradiation varied from 14 to 55%; TOC removal varied from 1.5 to 12.5%. Persistent degradation products were formed, and oxygen reactive species (1O2, oHO) showed to exhibit an important role during BPA degradation. The obtained quantum yield of BPA photolysis was 0.0075 mol BPA mol photons-1, determined from the experimental data through a mathematical model for BPA photolytic degradation versus time. With that aim a treatment system formed by a tubular reactor connected with a mixing tank with recirculation between them was considered; the tubular reactor was treated as a series of three associated continuous perfect mixing reactors (CSTR). Through statistical analysis of the experimental data for two responses (specific BPA degradation rate and BPA percent degradation at 120 minutes), the effects of the variables involved in BPA degradation (initial BPA concentration and specific rate of photon emission) were discussed.