Neste trabalho, estudou-se o efeito da adição de acetonitrila nas propriedades de micelas do detergente aniônico, dodecil sulfato de sódio (SDS), e do detergente catiônico, cloreto de hexadeciltrimetilamônio (CTACl). Medidas de condutividade foram utilizadas para determinar a concentração micelar crítica, cmc, e o grau de dissociação, α, das micelas em função da fração molar de acetonitrila, XAc. Medidas de supressão de fluorescência, resolvida no tempo, com pireno como sonda, foram utilizadas para determinar a influência de acetonitrila no número de agregação das micelas, N, e na dinâmica de migração de solutos entre as fases aquosa e micelar. Em baixas frações molares XAc < 0,2), a acetonitrila insere-se nas cavidades da água, quebrando parcialmente as pontes de hidrogênio da água com a formação de novas pontes de hidrogênio entre as moléculas de acetonitrila e as moléculas de água. Nesta faixa de concentração, ocorre um aumento da cmc e do α, acompanhada de uma diminuição de N. Observa-se também alterações na dinâmica da interação de contra-íons e co-íons supressores na micela. Assim, as micelas de SDS e CTACl formadas em misturas acetonitrila-água são menores, mais dissociadas e apresentam maior fluidez interna. Ao redor de XAc = 0,2, as misturas de água-acetonitrila tornam-se microheterogêneos com o aparecimento de microdomínios ricos em acetonitrila e microdomínios ricos em água. A proporção das regiões ricas em acetonitrila aumenta com o aumento da fração molar de acetonitrila, com apenas pequenas modificações das propriedades dos dois tipos de microdomínios. Em XAc > 0,2 a variação de cmc e de α com a XAc passa a ser menos acentuada, sugerindo que o detergente forma agregados preferencialmente nas regiões mais aquosas; a sonda fluorescente pireno começa sair das micelas durante o tempo de vida do estado excitado; e há claras mudanças na dinâmica de incorporação de íons nos agregados. / This work presents a study of the effect of added acetonitrile on the properties of the micelles of the anionic detergent sodium dodecylsulfate (SDS) and the cationic detergent hexadecyltrimethylammonium chloride (CTACl). Conductimetric measurements were employed to determine the critical micelle concentration, cmc, and the degree of counterion dissociation, α, of the micelles as a function of the mole fraction of added acetonitrile, XAc. Time resolved fluorescence quenching measurements with pyrene as probe were employed to determine the effect of acetonitrile on the micellar aggregation number, N, and the dynamics of solute migration between the micellar and aqueous phases. At low mole fractions (XAc < 0.2), acetonitrile inserts into the cavities present in liquid water, partially disrupting the hydrogen bonding of water, with formation of new hydrogen bonds between water and acetonitrile. In this range, both the cmc and α increase, while N decreases. The dynamics of incorporation of counterionic and coionic quenchers into the micelles is also altered. Thus, the SDS and CTACl micelles formed in these acetonitrile-water mixtures are smaller, more highly dissociated and internally more fluid than those in aqueous solution. Above XAc of ca. 0,2, acetonitrile-water mixtures become microheterogeneous, the solution containing microdomains rich in acetonitrile and microdomains rich in water. The proportion of acetonitrile-rich microdomians increases with increasing XAc, with only small changes in the properties of the two types of microdomains. Correspondingly, at XAc > > ca. 0.2: the variation of the cmc and α with XAc is much less pronounced, suggesting that the detergent forms aggregates preferentially in the aqueous-rich domains; the fluorescence probe pyrene begins to exit the micelles during its excited state lifetime; and there are distinct changes in the rate constants for the incorporation of ions into the micelles.

Le but de cette thèse est de sonder la flexibilité de NCp7 et de Δ(-)PBS, deux bio-molécules impliquées dans le second saut de brin de la transcription inverse du VIH. Deux stratégies expérimentales ont été mises en place. Un nouveau montage de spectroscopie ultra-rapide de fluorescence par down-conversion a été construit. Les dynamiques de quenching de la 2-aminopurine (2Ap), insérée en position 6, 8 et 10 de la boucle Δ(-)PBS ont pu être entièrement résolues à une résolution sub-ps. Pour chaque position, 4 temps de vie ont été révélés. Des mesures d'anisotropie confirment que les deux composantes < 5 ps sont liées à un empilement de la 2Ap avec les Guanines avoisinantes. Cet empilement est site-spécifique, prouvé par l'augmentation significative de leurs amplitudes lorsque la 2Ap est située près de la tige (position 10). La faible proportion de conformations reliées à un quenching collisionnel est significative de la faible exposition des 2Ap au solvant et de l'encombrement général de la boucle. La seconde approche avait pour but d'étudier l'effet du repliement du squelette protéique de [35-50] NCp7 autour de son atome de zinc par CID et par LID. Les spectres CID de la protéine nue sont expliqués par le modèle du proton mobile et une description détaillée d'un schéma de fragmentation spécifique autour du Tryptophane (Trp) a été soulignée, attribué une Lysine voisine. Un seul fragment issu de l'excitation à 266 nm a été identifié, son apparition entre en compétition avec les fragments CID du Trp. L'effet général du repliement autour du Zinc se traduit par une augmentation du taux de fragmentation autour du Trp et par une perte de spécificité pour le reste du spectre.Les flexibilités de Δ(-)PBS et NCp7 ont été respectivement évaluées par spectroscopie ultra-rapide de type down-conversion et par spectrométrie en phase gazeuse. La première méthode nécessite l'utilisation d'une sonde fluorescente non invasive, la 2-aminopurine (2Ap), placée en position 6, 8 et 10 de la boucle Δ(-)PBS. Notre résolution temporelle permet de résoudre entièrement les dynamiques locales de quenching et d'anisotropie de la 2Ap. Les composantes liées au quenching statique et quenching collisionnel ont été discriminées et révèlent les degrés d'empilement / encombrement locaux de la boucle. L'effet du repliement de [35-50] NCp7 autour de son atome de zinc a été étudié par CID et par LID à 266 nm. La protéine nue présente un interessant shéma de fragmentation autour du Tryptophane (Trp), exalté par la complexation avec le zinc, au prix une perte de spécificité pour le reste du spectre. Un seul fragment LID a été identifié, un mécanisme de sa formation est proposé. / This thesis aims to probe the flexibility of NCp7 and Δ(-)PBS, two biomolecules involved in the second strand transfer of the HIV's reverse transcription. We brought to the front two original experimental methods. A new ultrafast fluorescence down-conversion setup has been built, suitable for biological chromophore investigations. The quenching dynamics of 2-aminopurine (2Ap), site-mutated at the positions 6, 8 and 10 of Δ(-)PBS loop, were completely resolved under a ps scale. For each location, 4 decay times, were highlighted. Further anisotropy measurements confirmed that the two < 5 ps components correspond to stacking interactions of 2Ap with neighbouring Guanines. The site-specific aspect of the stacking were supported by a significant increase of their relative amplitudes when 2Ap were cloesly located to the stem (position 10). The minor portion of conformations involved with ps to ns collisional quenching suggests a low exposure of 2Ap towards the solvent as well as a general restriction of the loop. The second method planned to investigate the effet of the zinc-folding on [35-50] NCp7's peptidic backbone, thanks to CID and LID. The CID-generated spectra of the bare peptide were explained by the mobile proton model, and an exhaustive tryptophan (Trp) fragmentation pattern was described, mainly due to a neighbouring Lysin effects. Only one LID-fragment has been identified upon 266 nm excitation, probably created through a pathway competing with the generation of Trp fragments by CID. The main aspects related to zinc-folding are a general enhancement of the fragmentation ratios related to Trp and a loss of specificity for the remaining mass spectra parts.Δ(-)PBS et NCp7 has been respectively investigated by ultrafast down-conversion spectroscopy and gas-phase spectrometry. The first method implies the use of a non invasive fluorescent probe, named 2 aminopurine (2Ap), site mutated in position 6, 8 et 10 of the Δ(-)PBS loop. Our time resolution allows to fully depict the local quenching dynamics and anisotropy decays. The component related to static and collisional has been solved, thus describing different stacking degrees as well as local restrictions. The effect of [35-50] NCp7 folding around its zinc atom has been studied by CID and 266 nm LID. The bare protein displays an interesting fragmentation pathway around its Tryptophan (Trp), enhanced with zinc complexation, at the cost of a loss of specificity for the remaining mass spectra parts. Only one LID fragment has been identified, its occurence has been interpreted.

Neste trabalho, estudou-se o efeito da adição de acetonitrila nas propriedades de micelas do detergente aniônico, dodecil sulfato de sódio (SDS), e do detergente catiônico, cloreto de hexadeciltrimetilamônio (CTACl). Medidas de condutividade foram utilizadas para determinar a concentração micelar crítica, cmc, e o grau de dissociação, &#945;, das micelas em função da fração molar de acetonitrila, XAc. Medidas de supressão de fluorescência, resolvida no tempo, com pireno como sonda, foram utilizadas para determinar a influência de acetonitrila no número de agregação das micelas, N, e na dinâmica de migração de solutos entre as fases aquosa e micelar. Em baixas frações molares XAc &#60; 0,2), a acetonitrila insere-se nas cavidades da água, quebrando parcialmente as pontes de hidrogênio da água com a formação de novas pontes de hidrogênio entre as moléculas de acetonitrila e as moléculas de água. Nesta faixa de concentração, ocorre um aumento da cmc e do &#945;, acompanhada de uma diminuição de N. Observa-se também alterações na dinâmica da interação de contra-íons e co-íons supressores na micela. Assim, as micelas de SDS e CTACl formadas em misturas acetonitrila-água são menores, mais dissociadas e apresentam maior fluidez interna. Ao redor de XAc = 0,2, as misturas de água-acetonitrila tornam-se microheterogêneos com o aparecimento de microdomínios ricos em acetonitrila e microdomínios ricos em água. A proporção das regiões ricas em acetonitrila aumenta com o aumento da fração molar de acetonitrila, com apenas pequenas modificações das propriedades dos dois tipos de microdomínios. Em XAc &#62; 0,2 a variação de cmc e de &#945; com a XAc passa a ser menos acentuada, sugerindo que o detergente forma agregados preferencialmente nas regiões mais aquosas; a sonda fluorescente pireno começa sair das micelas durante o tempo de vida do estado excitado; e há claras mudanças na dinâmica de incorporação de íons nos agregados. / This work presents a study of the effect of added acetonitrile on the properties of the micelles of the anionic detergent sodium dodecylsulfate (SDS) and the cationic detergent hexadecyltrimethylammonium chloride (CTACl). Conductimetric measurements were employed to determine the critical micelle concentration, cmc, and the degree of counterion dissociation, &#945;, of the micelles as a function of the mole fraction of added acetonitrile, XAc. Time resolved fluorescence quenching measurements with pyrene as probe were employed to determine the effect of acetonitrile on the micellar aggregation number, N, and the dynamics of solute migration between the micellar and aqueous phases. At low mole fractions (XAc &#60; 0.2), acetonitrile inserts into the cavities present in liquid water, partially disrupting the hydrogen bonding of water, with formation of new hydrogen bonds between water and acetonitrile. In this range, both the cmc and &#945; increase, while N decreases. The dynamics of incorporation of counterionic and coionic quenchers into the micelles is also altered. Thus, the SDS and CTACl micelles formed in these acetonitrile-water mixtures are smaller, more highly dissociated and internally more fluid than those in aqueous solution. Above XAc of ca. 0,2, acetonitrile-water mixtures become microheterogeneous, the solution containing microdomains rich in acetonitrile and microdomains rich in water. The proportion of acetonitrile-rich microdomians increases with increasing XAc, with only small changes in the properties of the two types of microdomains. Correspondingly, at XAc &#62; > ca. 0.2: the variation of the cmc and &#945; with XAc is much less pronounced, suggesting that the detergent forms aggregates preferentially in the aqueous-rich domains; the fluorescence probe pyrene begins to exit the micelles during its excited state lifetime; and there are distinct changes in the rate constants for the incorporation of ions into the micelles.

Les protéines fluorescentes, comme la GFP (green fluorescent protein), sont des protéines naturellement fluorescentes qui sont utilisées pour leur rôle de marqueur, permettant de localiser des protéines dans les cellules et d'en suivre les déplacements. De nombreuses études expérimentales et théoriques ont été menées ces dix dernières années sur les protéines fluorescentes. De là, se forge une compréhension essentiellement qualitative du rôle de la protéine vis-à-vis de l’obtention ou non d’une émission radiative : il apparaît que la protéine permet la fluorescence en bloquant les processus qui la désactivent ; ces processus de désactivation sont très rapides et efficaces (à l'échelle de la picoseconde) dans le cas du chromophore seul, et ils sont bien identifiés comme étant des torsions autour des liaisons intercycles (tau et phi). Dans la protéine, la sensibilité des temps de vie de fluorescence à des mutations proches ou non du chromophore, à des modifications de pH ou de température laisse supposer un contrôle de la dynamique du chromophore par différents paramètres, sans qu’ils soient pour autant identifiés et mis en relation.Une étude de la dynamique de la protéine permettrait de faire la lumière sur les mécanismes responsables de ces phénomènes photophysiques pour lesquels une analyse structurale ne suffit pas. Cependant l'étude de la dynamique est limitée par la taille du système (>30 000 atomes), par l'échelle de temps des phénomènes photophysiques considérés (dizaine de nanosecondes) et par le fait que les deux torsions tau et phi sont fortement couplées dans l'état excité du chromophore. Ces trois facteurs excluent les méthodes de dynamique existantes aujourd'hui ; dynamique quantique (AIMD), dynamique mixte classique-quantique (QM/MD) et dynamique moléculaire classique (MD).Nous avons surmonté le problème par la modélisation de la surface d’énergie potentielle de torsion du chromophore à l’état excité basée sur des calculs quantiques de haute précision, par une interpolation des valeurs obtenues par une expression analytique appropriée en fonction des angles de torsion tau et phi et avec une précision suffisante pour reproduire des barrières de l’ordre de la kcal/mol, et enfin, par l’implémentation de cette expression analytique dans le programme parallèle AMBER. Une deuxième difficulté théorique concerne la simulation et l’analyse statistique d’événements peu fréquents à l’échelle de la nanoseconde, et dont on ne connait pas le chemin de réaction, ici les déformations de la protéine et du chromophore conduisant aux géométries favorables à la conversion interne. Grâce à ces développements et aux simulations qu'ils ont permises, nous avons réalisé la première modélisation de la désactivation non-radiative par conversion interne à l’échelle de la nanoseconde dans trois protéines fluorescentes différentes. L’analyse des dynamiques moléculaires classiques nous donne une évaluation quantitative des temps de vie de l’extinction de fluorescence, en accord avec les données expérimentales. Par ailleurs elle nous a permis d'identifier les mouvements moléculaires concertés de la protéine et du chromophore conduisant à cette extinction. De ces résultats, émerge une représentation plus complète du mécanisme qui libère la torsion du chromophore ou qui la déclenche : il peut venir d’un mouvement spécifique de la protéine, qui se produit à l’échelle de la nanoseconde, ou bien de plusieurs mouvements spécifiques, plus fréquents (rupture de liaisons hydrogène, rotation de chaînes latérales, dynamique d'agrégats d’eau), mais qui coïncident seulement à l’échelle de la nanoseconde. Ces mouvements spécifiques n’ont pas un coût énergétique important mais la nécessité de leur coïncidence crée un délai de l’ordre de quelques nanosecondes alors que dans le vide la torsion se produit en quelques picosecondes. Dans le cas des protéines étudiées, on a identifié en grande partie les mécanismes et les acides aminés qui sont impliqués. / Fluorescent proteins, like GFP (green fluorescent protein), are efficient sensors for a variety of physical-chemical properties and they are extensively used as markers in living cells imaging. These proteins have been widely studied both experimentally and theoretically the last decade. The comprehension of the protein's role in the regulation of the radiative emission is today essentially qualitative: it appears that the protein enables the fluorescence by blocking the processes that deactivates it; the deactivating processes are very quick and efficient (on the picosecond time scale) when the chromophore is isolated, and they are identified as being the torsions around the central bonds of the chromophore (tau and phi). The fluorescence lifetimes of a protein is very sensitive to mutations in the vicinity of the chromophore, to modifications in pH or in temperature. This seems to indicate a control of the dynamics of the chromophore by different parameters, that are not necessarily identified.A study of the dynamics of the protein would allow a deeper understanding of the mechanisms that are responsible for the fluorescence quenching. From a theoretical point of view, one is faced with three difficulties in this type of study: the size of the system (>30 000 atoms including a water box), the required time scale (tens of nanoseconds) and the fact that the torsions tau and phi are strongly coupled in the excited state of the chromophore. We must thus rule out the already existing dynamics methods: quantum dynamics (AIMD), mixed classical-quantum dynamics (QM/MD) and classical molecular dynamics (MD).We have overcome this problem by modeling the torsional potential energy surface of the chromophore in the first excited state trough high precision quantum calculations, by interpolating the energy values with an analytical fitting expression depending on the torsions tau and phi and with a precision high enough to reproduce barriers of the order of 1 kcal/mol, and lastly, by implementing this fitting expression in a parallelized version of the MD program AMBER. Another theoretical difficulty concerns the simulation and the statistical analysis of rare events on the nanosecond time scale without knowing the reaction path in advance, i.e. the deformations of the protein and of the chromophore leading to geometries where the internal conversion is favored. As a result of these developments and of the simulations they have enabled, we have been able to model, for the first time, the non-radiative deactivation by internal conversion at the nanosecond time scale in three different fluorescent proteins. The analysis of the classical molecular dynamics gives us a quantitative evaluation of the lifetime of the fluorescence extinction, in agreement with experimental results. In addition, it has allowed us to identify the concerted molecular movements between the protein and the chromophore leading to this extinction. A more complete representation of the mechanism that liberates or provokes the chromophore torsion emerges from these results: it could be a specific movement of the protein, that occurs on the nanosecond timescale, or several specific movements that occur more frequently (breakage of a hydrogen bond, rotation of side chains, dynamics of a water cluster), but that coincide only on the nanosecond time scale. These specific movements do not have a high energy cost but the need for them to coincide creates a delay of several nanoseconds compared to the chromophore torsion in vacuo which occurs after a few picoseconds. In the proteins we have studied (GFP, YFP and Padron), we have identified the principle components of the mechanisms and the amino acids that are implicated in this chromophore-protein interplay.