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Proteins modularity enhances the multi-functionality and versatility of proteins by providing such properties as multiple and various ligand-binding sites, increased ligand affinity through the avidity effect, and the juxtaposition of ligand-binding modules near catalytic domains. An NMR-based "dissect-and-build" approach to studying modular protein structure and function has proven very successful, whereby modules are initially characterized individually and then correlated with the overall function of a protein. We have used the dissect-and-build approach and NMR to study two modular protein systems. Chapter 2 details the NMR solution structure of the weak-lysine-binding kringle IV type 8 (KIV8) module from the apolipoprotein(a) (apo(a)) component of lipoprotein(a) was determined and its ligand-binding properties assessed. In vitro studies have demonstrated the importance of the apo(a) KIV7 and KIV8 modules in mediating specific lysine-dependent interactions with the apolipoproteinB-100 (apoB-100) component of LDL in the initial non-covalent step of lipoprotein assembly. Notable differences identified in the lysine binding site (LBS) of the KIV8 were deemed responsible for the differential modes of apoB-100 recognition by KIV7 and KIV8. In addition, the KIV8 structure has brought to light the importance of an RGD sequence at the N-terminus of the apo(a) KIV8 module, which may mediate important apo(a)-integrin interactions. In Chapters 3-6, structure-function studies of the CpGH84C X82 and the CpGH84A dockerin-containing modular pair were conducted to understand how the varying modularity unique to the C-terminal regions of the secreted multi-modular family 84 glycoside hydrolases influences the spreading of Clostridium perfringens. Identification of a CpGH84C cohesin module (X82), and the structural characterization of a dockerin-containing modular pair provides the first evidence for multi-enzyme complex formation mediated by non-cellulosomal cohesin-dockerin interactions. The formation of large hydrolytic enzyme complexes introduces a novel mechanism by which C. perfringens may enhance its role in pathogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-09-27 11:46:38.753

Recently, acremolin (4), a novel modified base, was isolated from a marine-derived fungus and claimed to possess a structure with a 1H-azirine moiety. It is shown now that the reported NMR data are not compatible with this antiaromatic heterocycle, which should be an extremely unstable compound. An isomeric, substituted N2,3-ethenoguanine is presented as a plausible alternative structure of acremolin that is consistent with all spectroscopic data. Thus, 1H-azirines keep their classification as very short-lived intermediates.

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity. / QC 20100924

Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general. / QC 20101025

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

Nuclear magnetic resonance (NMR) is a powerful experimental technique for probing the local environment of nuclei in materials. However, it can be difficult to separate the large number of interactions that are recorded in the resulting spectra. First-principles calculations based on quantum mechanics therefore provide much-needed support for interpreting experimental spectra. In this way, the underlying mechanisms recorded in experimental spectra can be investigated on an atomic level, and trends can be noted with which to guide the direction of future experiments. This thesis presents two cases in which first-principles calculations do just that. The first is an investigation of the perovskite structures of NaNbO₃, KNbO₃, LiNbO₃ and the related solid solutions of Na_xK_1-xNbO₃, K_xNa_1-xNbO₃ and Li_xNa_1-xNbO₃ in order to study how structural disorder affects their NMR parameters. The second investigation involves the calculation of the Knight shift in platinum, palladium and rhodium---in their elemental bulk forms and in a set of surface structures. The Knight shift is a systematic shift in the NMR frequencies of metallic systems. It arises from the hyperfine interaction between the nuclear spins and the spins of the unpaired conduction electrons. When calculating the Knight shift, it is found that the Brillouin zone must be very finely sampled. A discussion of core polarisation is also presented. This is the polarisation of core electrons as a result of their interaction with valence electrons. In the case of Curie paramagnets, core polarisation can have a significant effect on the calculation of hyperfine parameters.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-19T13:34:45Z No. of bitstreams: 5 gui.pdf: 4235660 bytes, checksum: a4da31a1b3421bf1fd546fc015611c65 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-20T10:39:27Z (GMT) No. of bitstreams: 5 gui.pdf: 4235660 bytes, checksum: a4da31a1b3421bf1fd546fc015611c65 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Made available in DSpace on 2015-02-20T10:39:27Z (GMT). No. of bitstreams: 5 gui.pdf: 4235660 bytes, checksum: a4da31a1b3421bf1fd546fc015611c65 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) Previous issue date: 2013-12-17 / Realizou-se a s?ntese de doadores e receptores glicos?dicos in?ditos que foram utilizados numa s?ntese convergente de um precursor da adenofostina A. Igualmente, a converg?ncia das sintonas tinha como prop?sito principal o estudo de rea??es de glicosila??o, ou seja, foram explorados dois m?todos de forma??o de liga??es glicos?dicas: o m?todo do tricloroacetoimidato e o m?todo de isomeriza??o do doador glicos?dico al?lico. A prepara??o dos aceptores e doadores glicos?dicos envolveu sequ?ncias de prote??o e desprote??o de grupos hidroxila a partir da adenosina e da D-glicose, respectivamente, privilegiando-se a forma??o de doadores armados e a observa??o da estereoqu?mica no carbono anom?rico. Os an?meros ? e ? formados nesta sequ?ncia de s?ntese foram separados, mas somente os an?meros ? foram motivo de caracteriza??o pelos m?todos de an?lise usuais de espectrometria RMN 1D e 2D, de massas e no infravermelho. Dessa forma, foram sintetizados dois aceptores glicos?dicos (17) e (18) e cinco doadores glicos?dicos (9), (11?), (11?), (13?) e (13?), sendo todas as subst?ncias in?ditas. Dentre os ensaios de glicosila??o somente o m?todo do tricloroacetoimidato produziu o produto desejado (glicosila??o entre 11? e 17) cuja caracteriza??o estrutural encontra-se em andamento. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2012. / ABSTRACT This works discusses the synton?s synthesis of the, unpublished glycosidic donors and aceptors that have been used in a convergent synthesis of a precursor of adenophostin A. Furthermore, the convergence of syntons main purpose was to study the glycosylation, ie been explored two methods of forming glycosidic linkages, the method of trichloroacetimidate and method of isomerization of the allyl glycoside donor. The preparation of donors and acceptors glycosidic sequences involving protection and deprotection of hydroxyl groups from adenosine and D-glucose, respectively, favoring the formation of armed donors and observation of stereochemistry at anomeric carbon. The and anomers formed in this synthesis sequence were separated, but the only reason anomers were characterization by usual methods of analysis of 1D and 2D NMR spectroscopy, mass and infrared. Thus, we synthesized two glycoside acceptors (17) and (18) and five donor glycoside (9), (11), (11), (13) and (13), all substances are novel. Among the glycosylation assays only the method of tricloroacetimidate gave the desired product (glycosylation between 11 and 17) that is the object of characterizing and whose data has not yet been completed and conclusive.

L’oxaliplatine, anticancéreux utilisé pour le traitement du cancer colorectal, est responsable d’une neurotoxicité périphérique dose-limitante affectant une grande majorité de patients. La neurotoxicité de l’oxaliplatine se présente sous deux formes : une forme immédiate, se traduisant par des paresthésies transitoires, et une forme retardée et cumulative, caractérisée par l’apparition d’une neuropathie périphérique douloureuse fortement invalidante. A l’heure actuelle, la prise en charge des douleurs neuropathiques est souvent incomplète, principalement à cause du manque de traitements efficaces et bien tolérés. Dans ce contexte, il existe un réel besoin d’innovation thérapeutique pour améliorer le traitement de ces neuropathies, nécessitant au préalable une meilleure compréhension de leur physiopathologie. La première partie de ce travail porte sur l’évaluation de l’effet d’une alimentation sans polyamines sur l’apparition et la chronicisation de la neurotoxicité de l’oxaliplatine chez le rat. En effet, en modulant positivement la sous-unité NR2B des récepteurs NMDA, les polyamines alimentaires pourraient faciliter la sensibilisation douloureuse. Un régime sans polyamines a permis de prévenir l’hypersensibilité thermique et mécanique induite par l’oxaliplatine. Bien que ces symptômes nociceptifs ne soient pas associés à une augmentation de l’expression de la sous-unité NR2B au niveau spinal, l’ifenprodil (antagoniste NR2B spécifique) permet d’en diminuer l’intensité de manière dose-dépendante. Enfin, une étude métabolomique réalisée par spectroscopie RMN du proton a montré que le régime sans polyamines permettait de réguler la neurotransmission excitatrice (glutamate) au niveau de la corne dorsale de la moelle épinière des animaux, expliquant ainsi son effet antalgique. Dans un deuxième temps, nous nous sommes intéressés aux mécanismes supraspinaux impliqués dans la neuropathie chronique induite par l’oxaliplatine, à l’aide d’une approche métabolomique par 1 H-RMN HRMAS. Cette étude a révélé d’importantes modifications métaboliques cérébrales chez les animaux traités par oxaliplatine, notamment une augmentation de la choline dans le cortex insulaire postérieur corrélée de manière significative aux seuils douloureux. Une analyse transcriptomique et pharmacologique a permis de mettre en évidence une implication de la neurotransmission cholinergique dans cette structure. Le ciblage pharmacologique de cette neurotransmission pourrait représenter une stratégie potentiellement intéressante pour le développement de nouveaux traitements antalgiques. L’ensemble de ces résultats expérimentaux a permis l’identification de nouvelles pistes pour la compréhension et le traitement des douleurs neuropathiques chimio-induites. Dans une perspective de recherche translationnelle, ces deux approches précliniques sont en cours de transposition dans des protocoles de recherche clinique. Un essai clinique de phase II (NEUROXAPOL, NCT01775449) a débuté afin de confirmer l’intérêt d’un régime appauvri en polyamines chez des patients recevant une chimiothérapie à base d’oxaliplatine. Une seconde étude clinique (INSULOX) est actuellement en cours de préparation au CHU de Clermont-Ferrand afin de mesurer par IRM les concentrations en choline dans l’insula de patients souffrant de douleurs neuropathiques induites par oxaliplatine. / Oxaliplatin, an anticancer drug used for the treatment of colorectal cancer, is responsible for a dose-limiting peripheral neurotoxicity in the majority of treated patients. This neurotoxicity appears with two components: a rapid-onset acute neurotoxicity manifesting as transient paresthesias and cold-induced dysesthesias; and a late-onset cumulative neurotoxicity characterized by the development of a painful chronic neuropathy. To date, the management of chemotherapy- induced neuropathic pain is still challenging because of the lack of effective treatments. In this context, a better understanding of the pathophysiological mechanisms underlying this neurotoxicity could lead to the identification of new therapeutic targets. Firstly, we aimed to assess the preventive effect of a polyamine deficient diet on the development of oxaliplatin-induced acute neurotoxicity. Exogenous polyamines, by positively modulating spinal NR2B-containing NMDA receptors, could facilitate pain sensitization. This study has shown that a polyamine deficient diet for 7 days totally prevented oxaliplatin-induced acute cold and mechanical hypersensitivity in rats. Although we observed no change in spinal NR2B expression or phosphorylation, intrathecal ifenprodil (a specific NR2B antagonist) reduced oxaliplatin-induced allodynia in a dose-dependent manner. Finally, proton NMR spectroscopy- based metabolomic analysis has revealed a regulation of spinal glutamate neurotransmission as the most likely mechanism underlying the preventive effect of the diet. Secondly, the metabolic variations associated with oxaliplatin-induced chronic neuropathy were assessed at the supraspinal level using a 1 H-NMR HRMAS-based metabolomic approach. Among the neurochemical changes evidenced in this study, we observed a significant increase in choline within the posterior insular cortex, significantly correlated with the mechanical pain thresholds. A transcriptomic and pharmacological approach have revealed an implication of cholinergic neurotransmission in this brain area. Targeting the cholinergic system using centrally active muscarinic agents could represent an interesting strategy for the treatment of oxaliplatin- induced neuropathic pain. These experimental results led to the identification of new molecular targets for the comprehension and the treatment of chemotherapy-associated painful neuropathy. In a translational approach, these preclinical data will be extended to the clinical setting. A phase II clinical trial (NEUROXAPOL, NCT01775449) is undergoing to confirm the therapeutic interest of a polyamine free diet in patients receiving oxaliplatin. A second clinical project (INSULOX) aiming at assessing the choline concentrations in the insula of patients suffering from oxaliplatin-induced neuropathy is in preparation.

L’imagerie par résonance magnétique est une technique non invasive et non ionisante permettant de caractériser la structure anatomique des tissus biologiques mous via la localisation des signaux de résonance magnétique nucléaire (RMN) des molécules mobiles. Cependant, l’application de l’IRM pour l’étude des matériaux rigides reste difficile dû aux forts élargissements des raies de résonances inhérents aux matériaux solides qui diminuent la résolution et le rapport signal sur bruit des images obtenues par encodage fréquentiel.La rotation à l’angle magique (MAS) permet de moyenner efficacement les interactions anisotropes de l’état solide par une rotation de l’échantillon, réduisant ainsi la largeur des raies de résonance. Dans ce manuscrit la possibilité de combiner la rotation à l’angle magique et l’IRM pour effectuer de la micro-imagerie multidimensionnelle et multi-nucléaire (¹H, ³¹P, ²⁷Al et ⁵¹V) à très haut champ magnétique (17,6 T) de matériaux solides avec une résolution et un rapport signal sur bruit largement supérieur à ceux obtenus en condition statique est démontrée. Une large gamme de matériaux (polymères, céramiques et tissus calcifiés biologiques) a été étudiée. Des images avec une résolution comprise entre 30 et 300 μm ont pu être obtenues pour des fréquences de rotation MAS allant jusqu’à 20 kHz, en utilisant des séquences IRM d’écho ou à temps d’écho nul. La possibilité d’utiliser un schéma de sous-échantillonnage associé à des algorithmes de reconstruction est aussi abordée.L’utilisation de séquences de RMN solide tels que la polarisation croisée pour augmenter le contraste et ainsi mettre en évidence des variations physico-chimiques localisées dans des tissus calcifiés biologiques est aussi démontrée. L’utilisation de gradients de champ magnétique pulsés combinés à la rotation à l’angle magique rend aussi possible la spectroscopie RMN de haute résolution localisée spatialement. Cette méthode a été utilisée pour étudier in vivo et dans chacun des segments du corps le métabolome de drosophiles modèles de pathologies neurodégénératives. / Magnetic Resonance Imaging (MRI) is a non-invasive and non-ionizing powerful tool widely used to characterize the structure and function of biological soft tissues through the localization of the NMR signal of mobile species. In contrast, the application of MRI in rigid solids remains challenging as they usually exhibit strong line broadening which decreases both the sensitivity and the resolution obtained with frequency encoding and short transverse relaxation time prohibiting the use of echo MRI sequences.Magic Angle Spinning (MAS) provides an efficient averaging of the an isotropic interactions in the solid statethrough a macroscopic rotation of the sample and allows obtaining narrow resonances. In this manuscript,we show the potentialities of combining MAS and MRI to carry out multi-nuclei (¹H, ³¹P, ²⁷Al or ⁵¹V) multidimensional micro-imaging in rigid solids, at very high magnetic field (17.6 T), with greatly improved SNR and spatial resolution when compared to static conditions. This is exemplified on a wide range of materials(polymers, oxide ceramics, biomaterials and hard tissues) for which the use of MAS at frequencies up to 20 kHz with spin-echo or Zero Echo Time (ZTE) MRI sequences allow obtaining images with spatialre solutions ranging from 30 to 300 μm. It is also demonstrated that solid state NMR sequences such as Cross-Polarization (CP) can be employed to enhance contrast and to further depict spatially localized chemical variations in bones and related materials. The possibility of using under sampled acquisition scheme with reconstruction algorithms is also addressed.The combination between pulsed field gradients and MAS also offers the possibility to perform high resolution localized spectroscopy. This methodology is used to study, in vivo and at the organ level, the metabolism of neuro degenerative pathologies drosophila models.