Le travail présenté dans le mémoire apporte une contribution dans le domaine de la synthèse et de la caractérisation structurale, spectroscopique et thermodynamique de ligands macropolycycliques dérivés du 5,12-dioxocyclame (L1). <br />La première partie est consacrée à l'étude du ligand L1, de son dérivé N-méthylé L2 et d'un récepteur ditopique constitué de deux unités dioxocyclamiques maintenues face-à-face par des ponts m-xylyle (L3). Des titrages potentiométriques ont mis en évidence la protonation coopérative des deux diamides monocycliques et un phénomène d'allostérie induit par le proton dans le cas du macrotricycle L3. Les complexes de nickel(II) et de cuivre(II) de géométrie plan-carrée ont été isolés et caractérisés par diffraction des rayons X, spectroscopies IR, UV-vis et RPE. La formation de liaisons hydrogène intermoléculaires entre les atomes d'oxygène amidiques et les molécules d'eau cocristallisées affectent l'énergie des orbitales d du cuivre. <br />La seconde partie relate la synthèse et les études physico-chimiques à l'état solide et en solution de macrobicycles possédant une anse o-xylyle (L4), m-xylyle (L5 et L7) ou pyridyle (L6). Leurs propriétés structurales et acido-basiques sont gouvernées par la formation de liaisons hydrogène intramoléculaires qui impliquent les amines tertiaires dont la protonation est lente à l'échelle de temps de la spectroscopie RMN. Les complexes de cuivre(II) correspondants ont été étudiés par diffraction des rayons X et par spectroscopies IR, UV-vis et RPE. Les ligands L4, L5 et L7 coordinent l'ion Cu2+ selon une géométrie plan-carrée déformée tandis que le ligand pentadentate L6 conduit selon le pH à la formation réversible d'un complexe tétra- ou pentacoordiné. La structure cristallographique du composé [Cu(L7)](ClO4)2 a montré que les contraintes stériques imposées par la cage moléculaire rigide induisent une déformation du noyau benzénique de l'anse et une interaction entre le métal et le cycle aromatique.

<p> This thesis contains an introduction to the surface chemistry of minerals in aqueous environment, and a summary of five manuscripts concerning adsorption reactions at the surfaces of nano-sized gibbsite (α-Al(OH)3), amorphous silica and kaolinite.</p><p> Nano-sized gibbsite was synthesized and thoroughly characterized using X-ray diffraction, high-resolution transmission electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. The adsorption of protons and the development of charge at the surfaces were studied using high precision potentiometry and zeta potential measurements. The results showed that singly coordinated surface sites at the particle edges protonate/deprotonate, while ion pairs with the medium ions are formed at doubly coordinated surface sites at the basal planes. This ion pair formation is a slow reaction, requiring long equilibrium times.</p><p> The adsorption of o-phthalate, maleate, fumarate, malonate and oxalate onto gibbsite surfaces was studied using Fourier transform infrared spectroscopy, zeta potential measurements, adsorption measurements and theoretical frequency calculations. All ligands were found to form outer-sphere complexes at the basal planes. Significant amounts of inner-sphere complexes at the particle edges were found for malonate and oxalate only. The observed adsorption was described using surface complexation models.</p><p> The proton reactions at the surface of amorphous silica were described using a two-site model. XPS indicated that Na+ is accumulated in the vicinity of the surface. Proton reactions at kaolinite surfaces were explained using a nonelectrostatic model, assuming that only the aluminol and silanol sites at the particle edges are reactive. Extensive modeling provided support for this assumption. </p>

Estrogen receptor α (ERα) is a member of a family of ligand-modulated transcription factors that have come to be known as nuclear receptors. ERα mediates the action of estrogens and plays an integral role in a wide range of physiological processes ranging from embryonic development and morphogenesis to reproduction to cardiovascular health. Not surprisingly, malfunction of the estrogen system is associated with a host of pathological conditions such as osteoporosis, heart disease and most notably breast cancer. Essential to its functioning as a transcription factor are specific protein-DNA interactions which are mediated by the binding of the DNA-binding (DB) domain of ERα to particular DNA sequences located within target gene promoters called estrogen response elements (EREs). Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry coupled with molecular modeling and semi-empirical analysis, I provide new insights into the ERα-DNA interaction in thermodynamic and structural terms. My data show that the binding of the DB domain of ERα to DNA is coupled to protonation at two specific amino acids, H196 and E203. Protonation of these residues is non-trivial and is required for high affinity binding. Amino acid sequence alignment of the DB domains of the NR family suggests that this may be a hallmark feature common to the functioning of all nuclear receptors. Furthermore, I demonstrate that the DB domain can tolerate all single nucleotide substitutions within the ERE and bind in the physiologically relevant nanomolar to micromolar range. Comparative thermodynamic analysis reveals that the DB domain binds to these ERE sequences utilizing a considerable range of energetic signatures such that any one thermodynamic component of binding is not predictive of associated affinity. In addition, it is shown that nucleotide substitution results in significant changes in secondary and three-dimensional features of the oligonucleotides and may impact binding affinity. Finally, I demonstrate that the zinc-finger of the DB domain of ERα is relatively promiscuous and can accommodate several heavy-metal divalent cations. Other than zinc, only DB domains reconstituted with cobalt, cadmium and mercury were capable of binding DNA. Incorporation of the metals resulted in a wide range of CD spectroscopic features which were found not to be predictive of DNA binding capacity. Thus, isostructure does not equate to isofunction in the case of metal reconstituted DB domain of ERα. This analysis suggests that metal coordination is not likely to be required for domain folding, but rather is required to bind DNA. Taken together, this thesis provides novel insights into the physicochemical basis of a key protein-DNA interaction essential to human health and disease. My studies bear the potential to impact the development of novel therapies harboring greater efficacy coupled with lower toxicity for the treatment of disease.

This thesis contains an introduction to the surface chemistry of minerals in aqueous environment, and a summary of five manuscripts concerning adsorption reactions at the surfaces of nano-sized gibbsite (α-Al(OH)3), amorphous silica and kaolinite. Nano-sized gibbsite was synthesized and thoroughly characterized using X-ray diffraction, high-resolution transmission electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. The adsorption of protons and the development of charge at the surfaces were studied using high precision potentiometry and zeta potential measurements. The results showed that singly coordinated surface sites at the particle edges protonate/deprotonate, while ion pairs with the medium ions are formed at doubly coordinated surface sites at the basal planes. This ion pair formation is a slow reaction, requiring long equilibrium times. The adsorption of o-phthalate, maleate, fumarate, malonate and oxalate onto gibbsite surfaces was studied using Fourier transform infrared spectroscopy, zeta potential measurements, adsorption measurements and theoretical frequency calculations. All ligands were found to form outer-sphere complexes at the basal planes. Significant amounts of inner-sphere complexes at the particle edges were found for malonate and oxalate only. The observed adsorption was described using surface complexation models. The proton reactions at the surface of amorphous silica were described using a two-site model. XPS indicated that Na+ is accumulated in the vicinity of the surface. Proton reactions at kaolinite surfaces were explained using a nonelectrostatic model, assuming that only the aluminol and silanol sites at the particle edges are reactive. Extensive modeling provided support for this assumption.

Ziel dieser Arbeit war die Untersuchung des Hydratationsverhaltens von alkylsubstituierten Aminen und deren korrespondierenden Ammoniumionen mit Hilfe von Car-Parrinello-Moleküldynamik Simulationen (CPMD). Dabei konnten Fragestellungen hinsichtlich der Koordinationszahlen, des Lösungsmittelaustausches und der molekülspezifischen pKB-Werte beantwortet werden. Des Weiteren lässt die Arbeit Aussagen über einen Zusammenhang von Hydratation und anormaler Basiszitätsreihenfolge der Amine zu und ermöglicht zusätzlich die Beschreibung von Systemen mit mehreren Aminfunktionen durch die Anwendung der gewählten Bedingungen. Zur Simulation des Hydratationsverhaltens wurden Wasser enthaltende Lösungsmittelboxen erstellt, bei denen das zu untersuchende Molekül zentral angeordnet war. Nach dem Ausschluss dimensionsabhängiger Effekte durch die Equilibrierung auf Grundlage einer kraftfeldbasierten Moleküldynamik, konnte eine geeignete Boxgröße bestimmt werden, die neben der Berechnung der ersten Hydratationssphäre auch die Beschreibung einer möglichen zweiten Hydratationssphäre erlaubt. Anhand der erhaltenen CPMD-Trajektorien aus den erfolgreichen Simulation wurden Verteilungsfunktionen berechnet. Der Vergleich dieser Ergebnisse mit experimentell bekannten Werten und Berechnungen an reinem Wasser zeigte, dass das System real vorliegende Bedingungen wiedergibt. Die ermittelten gNO(r) -Verteilungsfunktionen der Amine bzw. deren korrespondierender Ammoniumionen weisen signifikante Unterschiede auf und spiegeln einen grundsätzlich verschiedenen Aufbau der Hydratationssphären wieder. Dabei besitzen alle freien Amine eine starke Wasserstoffbrückenbindung unter Einbeziehung des freien Elektronenpaares am Stickstoffatom, während sich an den Aminwasserstoffatomen nur sehr schwache Wechselwirkungen beobachten ließen. Abgesehen vom Trimethylamin sind die Hydratationssphären der Amine wenig strukturiert und der Wasseraustausch zwischen erster und zweiter Hydratationssphäre verläuft sehr schnell. Im Gegensatz dazu ist die Umgebung der Ammoniumionen stark strukturiert. Alle Ammoniumwasserstoffatome sind in Wasserstoffbrückenbindungen einbezogen und es existiert ein vergleichsweise langlebiger Käfig aus Wassermolekülen um das Ammoniumion. Zusätzlich befindet sich zeitweise ein weiteres, wesentlich mobileres Wassermolekül in der ersten Hydratationssphäre, das in der Lage ist einen Wasseraustausch einzuleiten. Dabei konnten für das Ammoniumion und das Methylammoniumion konkrete Mechanismen des Wasseraustausches bestimmt werden. Die Berechnung der pKB- bzw. pKA -Werte für die untersuchten Systeme war ein weiterer Bestandteil der Arbeit. Dazu wurde ein statistischer Ansatz zur Ermittlung der freien Energie herangezogen, bei dem die Mittelwerte der Verteilungsfunktionen verwendet werden, so dass die zu erwartende Genauigkeit eng mit der Simulationszeit verknüpft ist. Auf Grundlage von Strukturoptimierungen an Ammoniumionen, die mit wenigen Wassermolekülen umgeben waren, konnten unter Variation der NH-Bindungslängen Energiegradienten ermittelt werden, die eine Aussage über die möglichen Übergangszustände bei der Deprotonierung lieferten. Als gute Näherung des Übergangszustandes kann demnach für alle Methylammoniumionen eine NH-Bindungslänge von 1,22 Ǻ in Betracht gezogen werden. Mit Hilfe dieser Bindungslänge wurde die Wahrscheinlichkeit der Deprotonierung für alle Ammoniumionen berechnet, wobei Simulationen bei denen ein spontaner Protonenübergang auftrat keine Berücksichtigung fanden. Die Ergebnisse der methylsubstituierten Amine zeigen eine gute Übereinstimmung mit den experimentellen Werten mit einer Abweichung von maximal +0,3 pK-Einheiten. Diese Abweichung entspricht in etwa 1,5 kJ/mol, was für theoretische Arbeiten einen sehr kleinen Fehler darstellt. Auf Grund der gewählten Bedingungen ist beim Ammoniumion die Wahrscheinlichkeit für die Deprotonierung unterschätzt wurden. Dadurch ist der entsprechende pKB -Wert kleiner als der experimentell ermittelte Wert. Im Verlauf der Arbeit konnte weiterhin gezeigt werden, dass das Verfahren und die gewählten Bedingungen auch für ethylsubstituierte Alkylamine und deren korrespondierende Ammoniumionen angewendet werden können. Die Genauigkeit der Ergebnisse hängt dabei hauptsächlich von der Simulationszeit ab. Ein entscheidender Punkt der Arbeit ist der Nachweis, das mit Hilfe dieser Moleküldynamik-Simulationen auch Moleküle mit mehreren Aminfunktionen berechnet werden können. Es ist somit möglich Differenzierungen in der Protonierung und Hydratation der einzelnen Aminfunktionen vorherzusagen.

Le transfert d'un analogue de lysine au travers de membranes de phospholipides a été étudié. Des simulations de dynamique moléculaire et des calculs d'énergie libre ont été conduits afin d'étudier l'évolution du pKa de cette molécule en fonction de sa position à l'intérieur de la membrane. Des grandeurs cinétiques telles que la perméabilité et les constantes de réaction associées au transfert ont montré que ce processus est susceptible de ce produire sur des échelles de temps appartenant à la ms. La comparaison de ces grandeurs dans des membranes constituées d'étherlipides et de lipides avec des chaînes branchées a été faite par la suite. Les études ont montré une diminution de la perméabilité et une augmentation du temps de passage dans des membranes faites d'étherlipides. L'ajout de méthyles le long des chaînes carbonées augmentent également le temps de passage et la perméabilité mais de manière moins importante. Cependant, le pKa indique que la forme chargée ne peut être retrouvée aussi profondément que dans une membrane constituée de chaînes linéaires. Finalement, le transfert du peptide a été étudié en estimant des surfaces d'énergie libre multidimensionnelles. La coordination de l'amine avec les molécules d'eau dans sa première sphère d'hydratation et la projection de la distance par rapport au centre de la membrane a été étudié. De nouvelles possibilités pour le changement d'état de protonation du peptide sont apparues. Contrairement au cas 1D, la forme neutre peut apparaître déjà dans la région des têtes polaires pour une certaine valeur de la coordination. Ces valeurs, inférieures à celles du milieux aqueux, peuvent être atteintes à l'interface d'après les surfaces déterminées / The transfer of a lysine amino acid analog across phospholipid membrane models was investigated using molecular dynamics simulations. The evolution of the protonation state of this small peptide as a function of its position inside the membrane was studied by determining the local pKa by means of free energy calculations. Permeability and mean first time passage were evaluated and showed that the transferoccurs on the sub-ms time scale. Comparative studies were conducted to evaluate the changes in the local pKa arising from the differences in the phospholipid chemical structure. We compared hence the effect of the ether vs ester linkage of the lipid head group as well as the linear vs branched lipid tails. The study reveals that protonated lysine residues can be buried further inside ether lipid membrane than ester lipid membrane while branched lipids are found to stabilize less the charged form compared to their un-branched lipid chain counterparts. As a result, the permeability and the transfer rate across a membrane constituted by ether lipid was found to slower than in membranes constituted by esterified lipids. Finally, multidimensionnal free-energy surfaces for the transfer of the peptide in its both states, charged and neutral, were estimated. The coordination of the amine with the water molecules in its first hydration shell with the projection of the distance from the center of the membrane were used as reaction coordinates. New possibilities for the deprotonation reaction were found, the latter appearing closer to the headgroup region. This finding suggests that if the lysine analog were less coordinated by water molecule a deprotonation is possible in the headgroup region

This Master thesis investigates the interaction between the polysaccharide sodium hyaluronate (HA) and some amphiphilic molecules. It is known that the presence of the carboxylic group on HA and the aminogroup on the amphiphiles leads to electrostatic interaction between these two compounds. This supposal offers the possibility to physically modify HA and use it as a new type of a carrier of bioactive compounds, for example medicals. However, successful carrier of bioactive compound has to resist a certain value of ionic strength. The high-molecular weights HA (1.75MDa) and amphiphile lysine were chosen for the study of the influence of ionic strength on the system HA – amphiphile. Our results show that system HA – amphiphile system is suppressed even by low concentrate solution of electrolyte. Therefore the system was reinforced by protonation of the aminoacid. The results show, that the interactions were reinforced, nevertheless negative influence of chlorine anions had to be eliminated by lyophilization. The solutions with strengthened system HA – amphiphile were used for the research of ionic strength influence. The amphiphiles lysine, 6 - aminocaproic acid and arginine were selected for this study. The interactions were investigated by means of reometry and conductometry.

<p> AroA catalyzes a carboxyvinyl transfer reaction, forming enolpyruvyl shikimate 3-phosphate (EPSP) from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP). Upon extended incubation, it forms EPSP ketal by intramolecular nucleophilic attack of O4H on C2' of the enolpyruvyl group. EPSP ketal was previously proposed to form by non-enzymatic breakdown of the tetrahedral intermediate (THI) which had dissociated from AroA. In this study, EPSP ketal formed in the presence of excess AroA, which demonstrated that it was formed in the active site. This eliminated non-enzymatic THI breakdown as its source, and demonstrated that AroA forms either a discrete EPSP cationic intermediate, or cl transition state with high cationic character. The pH dependence of non-enzymatic EPSP hydrolysis was examined in order to understand the intrinsic reactivity of the enolpyruvyl group. Acid catalysis accelerated EPSP hydrolysis> 10^8-fold. These results provide evidence for enolpyruvyl activation through protonation at C3', forming an unstable cationic intermediate, or a highly cation-like transition state. The incorporation of 2H into EPSP from solvent 2H20 during AreA-catalyzed hydrolysis was much slower than the hydrolysis rate, in the absence of inorganic phosphate in the reaction. This demonstrated that KIEs on AroA-catalyzed EPSP hydrolysis, when they are measured in the future, will reflect protonation of EPSP. A method was developed for KIE measurements on acid-catalyzed EPSP hydrolysis, which showed good reproducibility and no buffer dependence. Further experiments need to be completed on the acid-catalyzed KIEs and enzyme-catalyzed KIEs, followed by transition state analysis. This will precisely define the transition state structure of the enzyme-catalyzed EPSP hydrolysis reaction, and provide a good starting point for designing AroA inhibitors.</p> / Thesis / Master of Science (MSc)

Mis à part les liaisons hydrogène, d’autres interactions non covalentes participent dans les réseaux de reconnaissance ARN et ARN protéines. Parmi celles-ci, j’ai étudié les interactions oxygène-pi. Cette interaction prend la forme phosphate-pi dans les U turns et O4'-pi dans les motifs ARN-Z. Je propose une nouvelle classification des boucles de quatre nucléotides, décrivant les U turn et les Z turn à partir d’interactions oxygène-pi. De plus, les motifs "Z like" présents dans tous les ARN, sont aussi reconnus par certaines protéines immunologiques. Pour mieux comprendre les réseaux de reconnaissance biomoléculaire, nous avons examiné les interactions entre cations/anions et ARN. Nous avons trouvé de nombreuses erreurs dans les structures de la PDB et proposé des règles pour améliorer l'attribution d’espèces ioniques. Les résultats de cette thèse amélioreront notre connaissance des réseaux de reconnaissance biomoléculaire et aideront aux techniques de modélisation structurale des ARN. / Together with hydrogen bonds, uncommon non-covalent interactions are fundamental for recognition networks in RNA and RNA-protein systems. Among them, I focused on oxygen-pi stacking. This interaction takes the form of phosphate-pi within U-turns and of ribose O4’-pi within “Z-RNA” motifs. In that respect, a novel classification of tetraloops is proposed, defining U-turns and Z-turns based on their oxygen-pi stacking properties. Further, “Z-like” motifs are found to pervade small and large RNAs, being also a recognition pattern for immunology-related proteins. To better understand biomolecular recognition networks, we reviewed the binding of metal ions and anions within RNA, finding many examples of ions misattribution in PDB structures. We propose rules to avoid attribution errors. The results of this thesis will improve our knowledge and understanding of biomolecular recognition networks, as well as assist structural determination and structural modelling techniques of RNA systems.

Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr). ¶ The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrB•DHF•NADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB. ¶ Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a ‘minimalistic’ enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues. ¶ MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism. ¶ Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.