L’élaboration de nouveaux oligomères capables de mimer les propriétés des protéines naturelles est devenue un domaine de recherche très important, non seulement du point de vue structural mais aussi médicinal. Les peptides comportant des acides β-aminés ont été extensivement étudiés tandis que les recherches dans le domaine des γ-peptides sont plus récentes. Néanmoins, ces derniers montrent déjà des propriétés structurales uniques ainsi que des activités biologiques prometteuses.Dans notre laboratoire nous nous intéressons au développement d’une synthèse générale de nouveaux acides β,γ-diaminés à partir d’acides ∝-aminés naturels, notamment à la 3-déoxyaminostatine, et à leur utilisation dans la synthèse peptidique pour obtenir des peptides non-naturels afin d’étudier leurs propriétés conformationnelles. La stratégie de synthèse élaborée dans notre laboratoire et améliorée au cours de ces travaux a permis d’élargir la gamme des acides β,γ-diaminés. Deux diastéréomères cis et trans issus de la L-leucine, de la L- et D-phénylalanine ont été obtenus et engagés dans la synthèse de nouveaux peptides hybrides α/γ. Deux séries de peptides hybrides α/γ ont été étudiées. Les analyses structurales de la série comportant des acides ∝-aminés de configuration L montrent une capacité à adopter des structures secondaires bien définies, stabilisées par des liaisons hydrogènes intramoléculaires, impliquant notamment l’azote situé en β. De plus, une voie de synthèse vers un analogue d’un antibiotique naturel, le gramicidine S, a été proposée. Dans cet analogue, le motif D-Phe-L-Pro est remplacé par l’acide β,γ-diaminé issu de la D-phénylalanine. / The design of a new oligopeptides, capable to mimic the properties of natural proteins, is an important field not only for structural studies but also in the developpement of new efficient drugs. The peptides featuring β-amino acids have been extensively explored, whereas the research in γ-peptides is more recent. The studies of γ-peptides show their ability to adopt stable secondary structures and also to have a promising biological activity. Our laboratory is interested in the synthesis of new β,γ-diaminoacids.The aim of this work is a developpement of new synthetic route starting from different natural α -amino acids and to use obtained β,γ-diaminoacids to access novel unnatural peptides having specific conformational properties.The synthetic strategy, developed and optimized in our laboratory during this work, gives access to diastereomers cis and trans from L-leucine, L- and D-phenylalanine which were used in the synthesis of a new hybrid α/γ-peptides. The structural studies were performed on two series of hybrid α/γ-peptides consisting of β,γ-aminoacid from L-leucine and L- or D-α-amino acids. In the first case, the peptides are able to adopt stable secondary structures stabilized by intramolecular hydrogen bonds involving the nitrogen on the β-position. In addition, we present the synthesis of an analogue of gramicidine S, a naturally occuring antibiotic cyclic peptide. The dipeptide pattern D-Phe-L-Pro has been replaced with the β,γ-diaminoacid synthesized from D-phenylalanine.

Electrostatic interaction has long been proposed to be an important factor for stabilizing protein. Charge-charge interaction may especially be important to the thermostability of protein, as having more surface electrostatic interactions is one of the common structural features found in thermophilic proteins when compared to their mesophilic homologues. In order to quantitatively investigate the electrostatic contribution to protein stability, two complementary approaches, namely the double mutant cycle approach and pKa shift approach, were carried out. / In the double mutant cycle approach, the coupling free energies of two salt bridges (E6/R92 and K46/E62) and one a long range ion pair (E90/R92) were estimated by using circular dichroism, to find out the thermodynamic parameters of the protein model Thermococcus celer L30e and its charge-to-neutral mutants. It was found that the coupling free energy was temperature independent and was about 3 kJ mol-1 per salt bridge. By using a novel analysis of double mutant cycle of DeltaC p, it was also found that the interaction of salt bridge plays an important role in the reduction of DeltaCp. The temperature independency of coupling free energy and the effect of reducing DeltaCp could explain the general observation very well that thermophilic proteins have highly up-shifted protein stability curves is due to its elevated electrostatic interactions when compared with their mesophilic homologs. / In the pKa shift approach, the native state pKa values of acidic residues were obtained by fitting the side chain carboxyl 13C chemical shifts to microscopic model or global fitting of titrational event (GloFTE), whereas the denatured state pKa values were obtained by conventional pH titration of terminal protected 5-residue glycine-based model peptide. It was found that the surface charge-charge interactions, either attractive or repulsive, were strong and complicated because of the high surface charge density of T. celer L30e. However, the fact that most of the acidic residues have significantly downshifted native state pK a values indicated the surface charge distribution of T. celer L30e is optimized for stabilizing the protein. In addition, we have shown that temperature has negligible effect on pKa values in both native state and denatured state, therefore temperature can only marginally amplify the stabilizing effect in linear manner. / To overcome the unwanted crystallization problem of wild-type T. celer L30e in the low ionic strength neutral pH NMR conditions, which were essential for the pKa shift approach, a quintuple Arg-to-Lys variant was designed to dramatically improve the crystalline solubility, while the surface charges, as well as the structural, thermodynamic, and electrostatic properties, were conserved. It has also shown that electrostatic interaction played a critical role in crystallization at low ionic strength conditions, and arginine residue was especially important in crystal packing because of its high ability of forming salt bridges and hydrogen bonds. / Wild-type T. celer L30e has also shown to have no observable residual structure in the guanidine HC1-induced denatured state, indicating that denatured state of T. celer L30e should not have large effect on the overall protein stability. / Chan, Chi Ho. / Adviser: Kam Bo Wong. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 202-218). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

La dégradation intracellulaire des protéines est un processus fondamental qui a lieu dans tous les organismes, depuis les bactéries jusqu’aux êtres humains. La dégradation continuelle des protéines est nécessaire pour réguler les concentrations intracellulaires d’enzymes qui contrôlent toutes les réactions métaboliques, ainsi que le contenu général de toutes les autres protéines, en réponse aux modifications physiologiques. Un état d’équilibre dynamique se crée ainsi où la concentration intracellulaire d’une protéine peut être modulée aussi par des modifications de la vitesse de dégradation et de la vitesse de synthèse. Le travail présenté dans cette thèse porte sur le complexe d’activation du protéasome des cellules d’archaea (PAN). PAN est un complexe hexamerique énergie-dépendant découvert chez les archaeas et impliqué dans le dépliement des protéines substrat pour faciliter leur dégradation par le protéasome 20S. Toutes les études structurales du complexe PAN assemblé n’ont pas réussi à révéler la structure de la protéine à cause des difficultés rencontrées au niveau de la préparation et la stabilité des échantillons. La première partie du travail présenté a permis de déterminer une structure par Cryo-microscopie électronique et un modèle pseudo-atomique du complexe PAN hexamerique de Pyrococcus horikoshii. De plus, l’étude des différents états conformationnels du complexe induits par liaison du nucléotide ont permis de gagner plusieurs informations sur le mécanisme des unfoldases AAA+ et de proposer un mode d’action du complexe PAN. La seconde partie de l’étude a conduit à élucider la question sur la dynamique et les changements conformationnels des systèmes AAA+ unfoldases en général et du complexe PAN en particulier pendant le dépliement des protéines substrats. La méthode de variation de contraste en diffusion de neutrons aux petits angles (SANS) couplée avec de la spectroscopie de fluorescence appliqué à l’étude en temps réel du processus de dépliement de substrat par le système PAN de Methanococcus jannaschii a permis de révéler, pour la première fois, des mouvements de contraction de PAN pendant le dépliement du substrat induits par l’hydrolyse de l’ATP et suivis par une relaxation de la molécule à la fin du processus. Le mécanisme de dépliement par PAN semble être un mouvement de pompage péristaltique qui entraine un dépliement directionnel du substrat. L’ensemble de ce travail contribue à mieux comprendre la structure et le mécanisme d’action de la machine PAN dans les cellules et d’avoir une idée plus claire sur la dynamique fonctionnelle des complexes AAA+ à l’origine de leurs fonctions biologiques. / Intracellular protein degradation is a fundamental process occurring in all organisms, from bacteria to human. The continual degradation of proteins is necessary for regulating the intracellular levels of enzymes that control all metabolic reactions, as well as the general content of all other proteins, in response to physiological changes. A state of a dynamic equilibrium is created where the intracellular concentration of a protein can be modulated by changes in the synthesis rate as well as the degradation rate. The work presented in this thesis deals with the proteasome activating complex from archaeal cells (PAN). PAN is an energy dependent hexameric complex discovered in archaea and involved in the unfolding of protein substrates to facilitate their degradation by the 20S proteasome. All the previous structural studies on the assembled PAN complex have failed in revealing the structure of the whole complex because of the difficulties encountered during sample preparation and stabilization. In the first part of this work we determined a Cryo-electron microscopy structure and a pseudo-atomic model of the hexameric PAN complex from Pyrococcus horikoshii. In addition, the study of the different conformational states of the PAN complex induced by nucleotide binding helped in gaining several information about the AAA+ unfoldases mechanism and to propose a mode of action of the PAN complex. The second part of the study led to elucidate the question about the dynamic and the conformational changes of the AAA+ unfoldases in general and the PAN complex in particular. The method of contrast variation in Small Angle Neutron Scattering (SANS) coupled with online fluorescence spectroscopy applied to study, in real-time, the substrate unfolding process by the PAN complex from Methanococcus jannaschii allowed to reveal, for the first time, a contraction movements of PAN during substrate unfolding induced by ATP hydrolysis and followed by a relaxation of the molecule at the end of the process. The unfolding mechanism processed by PAN appears to be a peristaltic pumping motion that leads to a directional unfolding of the substrate. The whole work presented in this thesis contributes in understanding the structure and the mechanism of action of the PAN molecular machine inside the cells and to have a clearer idea about the functional dynamics of the AAA+ complexes at the origin of their biological functions.

Les polysaccharides amphiphiles possèdent des propriétés rhéologiques et interfaciales singulières dues aux interactions attractives entres les groupements hydrophobes greffés le long du squelette hydrophile. Leurs propriétés sont modulables suivant certains paramètres bien identifiés (nature des groupements hydrophobes, densité de greffage, etc.). Néanmoins, peu d’études s’intéressent à l’impact de la conformation du squelette sur le comportement de ces systèmes. L’objectif de ce mémoire est de déterminer l’influence de la rigidité du squelette sur les propriétés en solution d’un polysaccharide amphiphile à base de xanthane. En solution, ce polymère peut adopter deux conformations distinctes suivant les conditions opératoires : une forme ordonnée hélicoïdale rigide ou une forme désordonnée de type pelote flexible. Deux protocoles de greffage ont été développés afin de modifier le xanthane sous ces deux formes. Les xanthanes greffés sous forme ordonnée conservent une conformation hélicoïdale rigide, qui gouverne l’organisation globale des macromolécules. Au contraire, pour les polymères greffés sous forme désordonnée, l’organisation des chaînes est principalement contrôlée par les interactions attractives entre les groupements hydrophob / Hydrophobically modified polysaccharides show unusual rheological and interfacial properties in solution due to the self association of hydrophobic entities grafted onto their hydrophilic backbone. Their properties are tunable according to some well known parameters, such as the length of the hydrophobic moieties or the grafting density. However, very few studies deal with the influence of the backbone conformation on the properties of such systems in solution. Therefore, the objective of this work is to determine the impact of the backbone rigidity on the behavior of an amphiphilic polysaccharide based on xanthan. Indeed, in solution, this polymer can adopt two different conformations depending on the experimental conditions: an ordered, rigid helix or a disordered, flexible coil. Hydrophobic moieties have been grafted on xanthan either under its ordered and disordered conformation. Chains modified under the ordered conformation remain ordered and rigid after grafting and the overall properties are controlled by the high rigidity of the polymer backbone. On the contrary, macromolecules grafted under the disordered form show a more flexible conformation in solution. As a result, the organization of these derivatives is mainly controlled by the attractive interactions between the grafted moieties

Ce travail décrit l’incorporation de la δ-azaproline dans des pseudopeptides en vue d’analyses conformationnelles et applications biologiques. Le premier chapitre de ce manuscrit est consacré à l’élaboration d’un nouveau substrat de type Suc Ala-X-δazaPro-Phe-ρ-NA (où X est un acide aminé) capable de mesurer l’activité catalytique des PPIases. Ces enzymes ont déjà montré un grand intérêt dans la prévention des rejets de greffes et dans le traitement de la maladie d’Alzheimer car elles sont capables d’isomériser le lien X-Pro de la conformation cis vers la conformation trans. Le second chapitre décrit l’incorporation de la δ-azaproline dans des pseudotripeptides et l’étude de la réactivité des deux atomes d’azote de cet analogue. Il a pu notamment être mis en évidence que l’atome d’azote supplémentaire de la δ azaproline est beaucoup plus réactif que l’atome d’azote natif car l’encombrement stérique généré par le groupement Boc et l’oxydation spontanée de la liaison CHα NHδ représente un frein au bon déroulement du couplage peptidique. Finalement, la dernière partie de ce manuscrit est consacrée à l’analyse conformationnelle des pseudotripeptides synthétisés. Ces analyses ont pour but de déterminer l’influence d’un atome d’azote supplémentaire, en position δ du cycle de la proline, libre ou protégé par un groupement encombré sur la conformation du lien X-ΨPro. Les techniques spectroscopiques comme la RMN et l’IR ont permis de mettre en évidence que l’encombrement stérique généré par un groupement Boc en position δ avec la chaîne latérale de l’acide aminé en amont de cet analogue favorise la conformation cis par formation d’un pseudocycle en C10 / This work describes the incoporation the δ-azaproline in pseudopeptides for conformational analysis and biological applications. The first chapter of this thesis is devoted to the development of a new substrate of the type Suc Ala X δazaPro Phe ρ NA (where X is an amino acid) able to measure the catalytic activity of PPIases. These enzymes have shown great interests in the prevention of transplant rejection and in the treatment of Alzheimer's disease because they can interconvert the cis and trans isomers of X-Pro peptidic bond. The second section describes the incorporation of the δ-azaproline into pseudotripeptides in which the reactivity of both nitrogen atoms of this surrogate was investigated. It could be demonstrated in particular that the supplementary nitrogen atom of azaproline is much more reactive than the native one because of the steric hindrance generated by the Boc protecting group and the spontaneous oxidation of the CHα NHδ bond represent a huge problem in the peptidic coupling.Finally, the last part of this thesis is dedicated to the conformational analysis of pseudotripeptides. These analyzes are designed to determine the influence of a supplementary nitrogen atom, in δ position into proline’s cycle, which can be free or protected by a cluttered group on the conformation of the X-ΨPro bond. Spectroscopic techniques such as NMR and IR enabled to demonstrate that steric hindrance generated by a Boc group on δ position with the side chain of amino acid upstream this surrogate promotes cis conformation by formation of a ten-membered ring

Les polysaccharides amphiphiles sont constitués d’un squelette hydrophile sur lequel sont greffés des groupements hydrophobes. Ils possèdent des propriétés rhéologiques accrues dues à leur capacité d’auto-organisation en solution aqueuse mais également interfaciales dues à leurs propriétés d’adsorption aux interfaces eau/huile. Néanmoins, peu d’études s’intéressent à conférer ce type de propriétés à des polysaccharides complexes en termes de structure ou de conformation, comme le xanthane. En effet, ce polymère adopte en solution deux conformations distinctes selon les conditions expérimentales : une forme ordonnée hélicoïdale rigide et une forme désordonnée de type pelote flexible.Partant de ce constat, l’objectif de ce travail de thèse est de déterminer l’impact de la rigidité du squelette du xanthane modifié hydrophobiquement sur les propriétés amphiphiles en solution mais également sur les propriétés stabilisantes en émulsion.Il s’avère que la conformation adoptée par le polymère pendant le processus de greffage a un impact majeur sur ses propriétés rhéologiques : le xanthane modifié sous forme désordonnée possède un caractère associatif, contrairement au xanthane modifié sous forme ordonnée.De plus, le xanthane modifié hydrophobiquement est capable, sous certaines conditions, de former et de stabiliser des émulsions H/E, sans ajout de tensioactif moléculaire, en se partitionnant entre stabilisation de l’interface eau/huile et maintien de la viscosité de la phase aqueuse continue. / Amphiphilic polysaccharides are composed of a hydrophilic backbone grafted with hydrophobic moieties. They show specific rheological properties due to their capacity to self-associate in solution, but also interfacial properties due to their ability to adsorb at the water/oil interface. However, only few studies describe the chemical modification of complex heteropolysaccharides regarding their structure or their conformation, such as xanthan. Indeed, this polysaccharide can adopt in solution two distinct conformations depending on experimental conditions: an ordered rigid helix or a disordered flexible coil.The objective of this work is to determine the impact of the chain stiffness of hydrophobically modified xanthan on associative properties in solution, but also on stabilizing properties in emulsion.The conformation adopted by the polymer during the grafting procedure has a major impact on the rheological properties: xanthan modified under disordered conformation is an associative polysaccharide, contrary to xanthan modified under its ordered conformation.In addition, hydrophobically modified xanthan is able, under certain conditions, to form and stabilize O/W emulsion, without further addition of molecular surfactant, by partitioning between stabilization of the water/oil interface and viscosifying the continuous aqueous phase.

Paclitaxel was isolated from the bark of <i>Taxus brevifolia</i> in the late 1960s. It exerts its biological effect by promoting tubulin polymerization and stabilizing the resulting microtubules. Paclitaxel has become one of the most important current drugs for the treatment of breast and ovarian cancers. Studies aimed at understanding the biologically active conformation of paclitaxel bound on β–tubulin are described. In this work, the synthesis of isotopically labeled taxol analogs is described and the REDOR studies of this compound complexed to tubulin agrees with the hypothesis that palictaxel adopts T-taxol conformation. Based on T-taxol conformation, macrocyclic analogs of taxol have been prepared and their biological activities were evaluated. The results show a direct evidence to support T-taxol conformation. (+) Discodermolide is a polyketide isolated from the Caribbean deep sea sponge <i>Discodermia dissoluta</i> in 1990. Similar to paclitaxel, discodermolide interacts with tubulin and stabilizes the microtubule <i>in vivo</i>. Studies aimed at understanding the biologically active conformation of discodermolide bound on β–tubulin are described. In this work, the synthesis of fluorescent labeled discodermolide analogs is described and their biological activities were evaluated. Synthetic approaches to fluorescent labeled and isotopically labeled discodermolide analogs discodermolide are also described. / Ph. D.

Submitted in partial fulfilment for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / This study involved a series of molecular dynamics (MD) simulations applied to case studies of small and medium-size polypeptides to assess the thermodynamics of their folding characteristics. Peptide folding is a complex and vital phenomenon taking place in all living systems. Bioactive conformational structures of folded peptides need to be well characterized before using them in computer-aided drug design. The computational procedure was validated on the 10-residue long chignolin-like synthetic mini-protein (CLN025). For this peptide, replica exchange molecular dynamics (REMD) calculations were carried out in explicit and implicit solvents using the generalized Born (GB)/surface area (SA) approximation with different sets of force field parameters. Following this validation procedure, case studies of the folding conformations of peptides of different lengths including the 5-residue met-enkephalin, the 27-residue pituitary adenylate-activating polypeptide 27(PACAP27) and the 28-residue vasoactive intestinal peptide (VIP) were undertaken. The latter two peptides are multifunctional hormones that mediate diverse biological functions, such as the cell cycle, cardiac muscle relaxation, immune response, septic shock, bone metabolism, and endocrine function. Results obtained indicate that when explicit water, methanol and DMSO solvents were used, it appeared that methanol (MeOH) and dimethylsulphoxide (DMSO) afforded met-enkephalin the ability to form more intra-hydrogen bonds than water, producing type I and type III β-turn structures; thus enhancing the helical conformation of the peptide. MD trajectories of longer polypeptides (VIP and PACAP27) were also populated with type I and type III β-turns, which occurred consecutively; with α- and 310-helices occurring from the middle of each peptide towards the C-terminal. Characterization of implicit solvent results, reveal that these simulations have been able to reproduce the same type of conformers obtained by experimental NMR studies published in literature, which structurally resemble the native conformation of the bioactive peptides. These conformational structures will be applied as lead agents in computer-aided drug design. One of the major achievements of this study is the ability to optimize and validate the force field parameter sets to describe the thermodynamic properties of peptide systems in an unbiased manner, a non-trivial task for even the smallest of peptides. These findings re-affirm the notion that computational methods have matured enough to model dynamic biological phenomena such as peptide folding, a feat previously thought to be impossible.

For proteins the link between their structure and their function is a central tenet of biology. A common approach to understanding protein function is to ‘solve’ its structure and subsequently probe interactions between the protein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their entire structure fail to fold into a three-dimensional structure and yet they possess function. These so called intrinsically or inherently disordered proteins (IDP’s) or intrinsically disordered regions (IDR’s) constitute up to 40% of all expressed proteins. IDPs which have crucial roles in molecular recognition, assembly, protein modification and entropic chain activities, are often dynamic with respect to both conformation and interaction, so in the course of a protein’s ‘lifespan’ it will sample many configurations and bind to several targets. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. The solvent free environment of a mass spectrometer is ideally suited to the study of intrinsic interactions and how they contribute to structure. Ion mobility mass spectrometry is uniquely able to observe the range of structures an IDP can occupy, and also the effect of selected binding partners on altering this conformational space. This thesis details the technique of ion mobility mass spectrometry and illustrates its use in assessing the relative disorder of p53 protein. The tumour suppressor p53 is at the hub of a plethora of signalling pathways that maintain the integrity of the human genome and regulate the cell cycle. Deregulation of this protein has a great effect on carcinogenesis as mutated p53 can induce an amplified epigenetic instability of tumour cells, facilitating and accelerating the evolution of the tumour. Herein mass spectrometry provides a compelling, detailed insight into the conformational flexibility of the p53 DNA-binding domain. The plasticity of the p53 DNA-binding domain is reflected in the existence of more than one conformation, independent of any conformational changes prompted by binding. The in vacuo conformational phenotypes exhibited by common cancer-associated mutations are determined and the second-site suppressor mutation from loop L1, H115N, is probed whether it could trigger conformational changes in p53 hotspot cancer mutations. The structural basis of the binding promiscuity of p53 protein is investigated; of particular interest is the molecular interaction of the p53 N-terminus with the oncoprotein murine double minute 2, as well as with the antiapoptotic factor B-cell lymphoma-extralarge.

Cette étude est consacrée à l'analyse des propriétés électrostatiques et structurales des protéines diffractant à haute résolution. Les travaux se déclinent en deux aspects principaux qui sont, d'une part, l'analyse d'un point de vue fondamental des propriétés dérivées de leur distribution de charge des structures des protéines, et, d'autre part, l'application pratique des méthodes de la cristallographie haute résolution des macromolécules à deux enzymes.Grâce à une analyse structurale et électrostatique de la protéine PfluDING, le mode de fixation du phosphate a été mis en évidence à deux pH différents. En particulier, cette étude a permis de démontrer la présence d'une liaison hydrogène diffuse assurant un mode de fixation identique quelque soit le pH. La seconde enzyme étudiée est la protéine DFPase, capable de dégrader les organophosphorés. La structure de la DFPase obtenue par diffraction X haute résolution et la structure affinée contre des données neutroniques ont été comparées. L'analyse pointe les différences d'interprétation des deux modèles, et permet de proposer un nouveau mécanisme d'action.Les aspects plus fondamentaux de cette étude portent sur les éléments de structure secondaire des protéines : leurs propriétés électrostatiques en termes de moments électriques (moments dipolaires et quadripolaires des hélices et des feuillets), et le réseau des interactions (dont les liaisons H) assurant la cohésion des hélices ont été analysés. Il a été démontré comment certaines interactions entre liaisons peptidiques au sein d'hélices, classiquement représentées comme des liaisons hydrogène, devaient être considérés comme des contacts purement électrostatiques / This study is about the electrostatic and structural properties of proteins diffracting at high X-ray resolution. Two different aspects are tackled which are 1) the analysis of properties derived from their charge distribution, parting from a fundamental point of view and 2) the application of methods used in high resolution macromolecular crystallography to two enzymes of major interest.After the analysis of the structure and electrostatic properties of PfluDING protein, the binding mode of a phosphate ion, located in the active site, was elucidated at two different pH values. Particularly, this study demonstrates that a diffuse hydrogen bond assures the protonation state of the phosphate ion, which is thus identical at each pH value. The second enzyme studied is the proteine DFPase which is capable of hydrolysing nerve agents. A high resolution X-ray structure and a medium resolution neutron structure where compared. The analysis points out the differences between the two models and a new catalytic mechanism could be proposed.The more fundamental aspects of this study are about the secondary structural elements of proteins: their electrostatic properties in terms of electrostatic moments (dipole and quadrupole moments of helices and sheets) as well as the hydrogen bond network assuring the cohesion of helices have been analyzed. It has been shown that certain interactions between peptide units within helices, represented usually as hydrogen bonds, should actually be considered as pure electrostatic contacts