Détermination par résonance magnétique nucléaire de structures tridimentionnelles de peptides et de protéines (Structures tridimentionnelles de deux peptides transporteurs de fer chez les bactéries Gram-négatif.Etudes structurales et dynamiques de trois domaines du facteur humain de transcription/réparation TFIIH)

Les fonctions spécifiques des protéines résultent de leurs caractéristiques structurales et dynamiques. La spectroscopie de Résonance Magnétique Nucléaire (RMN) est l'une des principales techniques apportant ces deux types d'informations, tant pour de petits peptides que pour des protéines plus imposantes. Cependant, les stratégies d'études se différencient selon la taille des biomolécules. Durant mon travail de thèse, je me suis attaché à l'étude structurale par spectrométrie RMN de deux types de molécules intervenant dans deux contextes biologiques différents.Le premier contexte biologique abordé est celui du métabolisme du fer chez les bactéries Gram-négatif. Les études structurales de deux sidérophores peptidiques bactériens sont présentées : l'azoverdine d'Azomonas macrocytogenes ATCC 12 334 et la pyoverdine PaA de Pseudomonas aeruginosa ATCC 15 692. Le but de ces études est la compréhension du mécanisme de reconnaissance spécifique complexe métallique sidérophore/récepteur membranaire. Nous avons adapté à l'étude de ces peptides des méthodes utilisées sur les protéines. Ceci comprend une séquence de type HNCA utilisée en abondance naturelle de 13C et l'utilisation des couplages dipolaires résiduels (RDC). Nous avons également étudié le cas de plusieurs conformères en échange chimique intermédiaire.Le second contexte biologique abordé est celui de la transcription et de la réparation de l'ADN via l'étude structurale et dynamique de trois domaines de sous-unités du facteur humain de transcription/réparation TFIIH: MAT1, p44 et p62. La première partie décrit la détermination structurale du domaine RING C3HC4 de la sous-unité MAT1. La seconde partie présente une étude sur la dynamique et les propriétés d'échange métallique du domaine RING C8 de la sous-unité p44. Enfin, la troisième partie démontre l'apport des contraintes orientationnelles issues des RDC dans l'affinement des structures tridimensionnelles du domaine PH de la sous-unité p62.The specific functions of proteins result from their structural and dynamic features. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods which provides these two kinds of informations both for small peptides and for larger proteins. However the investigation strategie is different depends on the size of biomolecules. My thesis work is concerned with NMR structural studies of two kinds of molecules acting with various biological contexts.The first type of molecules studied are involved in the iron-transport process of Gram-negative bacteria. Structural studies of two peptidic bacterian siderophores are presented: azoverdin from Azomonas macrocytogenes ATCC 12 334 and pyoverdin PaA from Pseudomonas aeruginosa ATCC 15 692. The aim of these NMR studies is to understand the specific recognition mechanisms between metal-complexed siderophores and their membrane receptors. We adapted methods which are routinely used in protein NMR spectroscopy to the study of these peptides. These include 13C natural abundance HNCA pulse sequence and the use of residual dipolar couplings (RDC). We studied too case of intermediate time-scale chemical exchange between several conformers.The second type of molecules investigated are protein domains involved in DNA transcription and repair. The structural and dynamic characterisation of three domains of transcription / DNA repair human factor TFIIH subunits is described. Initially the solution structure of the RING C3HC4 domain of the MAT1 subunit was solved. Afterwards the dynamic behaviour and metal exchange properties of the RING C8 domain of the p44 subunit were investigated. Finally the contribution of orientational constraints issued from residual dipolar couplings in three-dimensional structure refinement of the PH domain of the p62 subunit is demonstrated.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The Three-Dimensional Structure of the Gallium Complex of Azoverdin, a Siderophore of Azomonas macrocytogenes ATCC 12334, Determined by NMR Using Residual Dipolar Coupling Constants

International audienceIn iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins

Solution Structure of the N-terminal Domain of the Human TFIIH MAT1 Subunit

International audienc

Solution and Solvent‐Free Stopper Exchange Reactions for the Preparation of Pillar[5]arene‐containing [2] and [3]Rotaxanes

International audienceAbstract Diamine reagents have been used to functionalize a [2]rotaxane building block bearing an activated pentafluorophenyl ester stopper. Upon a first acylation, an intermediate host‐guest complex with a terminal amine function is obtained. Dissociation of the intermediate occurs in solution and acylation of the released axle generates a [2]rotaxane with an elongated axle subunit. In contrast, the corresponding [3]rotaxane can be obtained if the reaction conditions are appropriate to stabilize the inclusion complex of the mono‐amine intermediate and the pillar[5]arene. This is the case when the stopper exchange is performed under mechanochemical solvent‐free conditions. Alternatively, if the newly introduced terminal amide group is large enough to prevent the dissociation, the second acylation provides exclusively a [3]rotaxane. On the other hand, detailed conformational analysis has been also carried out by variable temperature NMR investigations. A complete understanding of the shuttling motions of the pillar[5]arene subunit along the axles of the rotaxanes reported therein has been achieved with the help of density functional theory calculations

Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn2+-Cd2+exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved. © 2005 Elsevier Ltd. All rights reserved

Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn2+-Cd2+exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved. © 2005 Elsevier Ltd. All rights reserved

Quantification of Cholesterol Solubilized in Dietary Micelles: Dependence on Human Bile Salt Variability and the Presence of Dietary Food Ingredients

The solubility of cholesterol in bile salt (BS) micelles is important to understand the availability of cholesterol for absorption in the intestinal epithelium and to develop strategies to decrease cholesterol intake from the intestinal lumen. This has been the subject of intense investigation, due to the established relation between the development of diseases such as atherosclerosis and high levels of cholesterol in the blood. In this work we quantify the effect of BS variability on the amount of cholesterol solubilized. The effect of some known hypocholesterolemic agents usually found in the diet is also evaluated, as well as some insight regarding the mechanisms involved. The results show that, depending on the bile salt composition, the average value of sterol <i>per</i> micelle is equal to or lower than 1. The amount of cholesterol solubilized in the BS micelles is essentially equal to its total concentration until the solubility limit is reached. Altogether, this indicates that the maximum cholesterol solubility in the BS micellar solution is the result of saturation of the aqueous phase and depends on the partition coefficient of cholesterol between the aqueous phase and the micellar pseudophase. The effect on cholesterol maximum solubility for several food ingredients usually encountered in the diet was characterized using methodology developed recently by us. This method allows the simultaneous quantification of both cholesterol and food ingredient solubilized in the BS micelles even in the presence of larger aggregates, therefore avoiding their physical separation with possible impacts on the overall equilibrium. The phytosterols stigmasterol and stigmastanol significantly decreased cholesterol solubility with a concomitant reduction in the total amount of sterol solubilized, most pronounced for stigmasterol. Those results point toward coprecipitation being the major cause for the decrease in cholesterol solubilization by the BS micelles. The presence of tocopherol and oleic acid leads to a small decrease in the amount of cholesterol solubilized while palmitic acid slightly increases the solubility of cholesterol. Those dietary food ingredients are completely solubilized by the BS micelles, indicating that the effects on cholesterol solubility are due to changes in the properties of the mixed micelles

Unexpected aqueous UCST behavior of a cationic comb polymer with pentaarginine side chains

Thermoresponsive polymers, undergoing a reversible chemical or physical change using temperature as stimulus, attract increasing interest in particular as adaptable biomaterials. Except for zwitterionic polymers, fully charged polymers require the presence of specific ions to exhibit an upper critical solution temperature (UCST) in water. Herein, we report the discovery of an UCST in pure water for fully cationic comb polymers based on oligoarginine pendent grafts. These polymers were prepared using an original strategy based on solid-phase peptide synthesis of pentaarginine methacrylate-based macromonomer and its polymerization through reversible addition-fragmentation chain transfer. Despite their cationic nature, guanidinium groups from the arginine have the ability to self-associate at low temperature through hydrophobic interactions into stacked pair configuration defying the expected Coulomb interactions. These results pave the way to biomedical applications such as antimicrobial materials and drug delivery systems through the tuning of the polymer structure