Les protéines membranaires sont particulièrement riches en acides aminés aromatiques, tels que le tryptophane (W). On retrouve cette originalité dans beaucoup de peptides antimicrobiens et dans les protéines de fusion virales. La glycoprotéine de l'enveloppe de HIV-1, gp41, en est un exemple. Manifestement, les résidus W sont impliqués dans la perturbation des membranes et la formation des pores. L'objectif de ce travail est d'étudier le rôle des résidus W dans de telles activités en utilisant l'optique non linéaire. Pour cela, nous avons préalablement déterminé l'hyperpolarisabilité (le potentiel non linéaire) du W par la diffusion Hyper Raleigh (HRS). Puis nous avons montré une évolution de la réponse non linéaire de petits peptides synthétiques en fonction du nombre croissant de leurs résidus W. Ces résultats ont permis de suivre l'implication des tryptophanes de deux peptides K3W et gp41W, lors de leurs interactions avec des monocouches lipidiques à l'interface air-eau par la génération de second harmonique (SHG). D'autre part, l'influence de telles interactions sur la structure secondaire et l'orientation des peptides a été déterminée par le PM-IRRAS. Nous avons ainsi montré la cohérence entre les modifications du signal SHG, liées à des changements d'orientation des tryptophanes et celles des spectres de PM-IRRAS, dues à des changements d'orientation de la structure secondaire de gp41W / Membrane proteins are extremely rich in aromatic amino acids, like tryptophan (W). This particularity is found in many antimicrobial peptides and in several virus fusion proteins. An example of these fusion proteins is the HIV-1 envelop glycoprotein, the gp41. It is clear that the W residues are implicated in membrane perturbation and pore formation. The aim of this work was the investigation of the W residue role in such activities, using the nonlinear optic. First, we determined the W hyperpolarizabilité (nonlinear potential) by the Hyper Rayleigh Scattering (HRS). Then, the evolution of the nonlinear signal of small synthetic peptides, as function of the increasing number of their W residues, was demonstrated. These results allowed us to follow the W residue involvement of two peptides, K3W4 and gp41W, in the interaction with lipids monolayer at the air-water interface, using the second harmonic generation (SHG). The influence of such interaction in the peptide structure and orientation was determined using the PM-IRRAS. In conclusion, we showed the coherence between the SHG signal variation, due to the W orientation changes, and the PMIRRAS spectra modification, due to the gp41W helix orientation changes

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

TDO utilizes b-type heme as a cofactor to activate dioxygen and insert two oxygen atoms into free L-tryptophan. We revealed two unidentified enzymatic activities of ferric TDO from Ralstonia metallidurans, which are peroxide driven oxygenation and catalase-like activity. The stoichiometric titration suggests that two moles of H2O2 were required for the production of one mole of N-formylkynurenine. We have also observed monooxygenated-L-tryptophan. Three enzyme-based intermediates were sequentially detected in the peroxide oxidation of ferric TDO in the absence of L-Trp including compound I-type and compound ES-type Fe-oxo species. The Fe(IV) intermediates had an unusually large quadrupole splitting parameter of 1.76(2) mm/s at pH 7.4. Density functional theory calculations suggest that it results from the hydrogen bonding to the oxo group. We have also demonstrated that the oxidized TDO was activated via a homolytic cleavage of the O-O bond of ferric hydroperoxide intermediate via a substrate dependent process to generate a ferrous TDO. We proposed a peroxide activation mechanism of the oxidized TDO. The TDO has a relatively high redox potential, the protonated state of the proximal histidine upon substrate binding as well as a common feature of the formation of ferric hydroxide species upon substrate or substrate analogues binding. Putting these together, we have proposed a substrate-based activation mechanism of the oxidized TDO. Our work also probed the role of histidine 72 as an acid-base catalyst in the active site. In H72S and H72N mutants, one water molecule plays a similar role as that of His72 in wild type TDO. MauG is a c-type di-heme enzyme which catalyze the biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Its natural substrate is a monohydroxylated tryptophan residue present in a 119-kDa precursor protein of methylamine dehydrogenase (MADH). We have trapped a novel bis-Fe(IV) intermediate from MauG, which is remarkably stable. A tryptophanyl radical intermediate of MADH has been trapped after the reaction of the substrate with the bis-Fe(IV) intermediate. Analysis by high-resolution size-exclusion chromatography shows that MauG can tightly bind to the biosynthetic precursor and form a stable complex, but the mature protein substrate does not.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Artificial photosynthesis is often considered to have great potential to provide alternative, renewable fuels by harvesting, conversion and storage of solar energy. One promising approach is the development of modular molecular photocatalysts inspired by natural photosynthetic enzymes. The first part of this thesis deals with artificial mimics of the water oxidizing photosystem II composed of a chromophore and an electron relay as synthetic counterpart of the P680-TyrZ/His190 ensemble of photosystem II. Three ruthenium polypyridyl - imidazole - phenol complexes with varying position of a methyl group on the phenol ring (Ru-xMe) were synthesized and characterized by electrochemical and photophysical methods. As an improvement compared to earlier complexes the increased redox potential (~0.9 V vs. Ferrocene) of the phenol groups makes their function as an electron relay in a photocatalytic system for water oxidation thermodynamically possible. Time-resolved absorption studies revealed fast intramolecular electron transfer (<5-10 µs in aprotic solvent and <100 ns in water) despite the low driving force and the importance of the hydrogen bond between the phenol and the imidazole group was put in evidence. Slight differences between the three Ru-xMe complexes and investigation of the effect of external bases allowed to derive a mechanistic picture in which the imidazole is involved in a "proton domino" reaction. Accepting the phenolic proton upon ligand oxidation (within the H-bond) renders its second nitrogen site more acidic and only deprotonation of this site pulls the overall equilibrium completely towards oxidation of the ligand. Another part of this thesis comprises a chromophore-tryptophan construct synthesized using a click chemistry approach. Light-induced oxidation of Trp in this Ru-tryptophan complex was shown to follow ETPT mechanism. Depending on the pH conditions tryptophan radicals, either Trp* or TrpH*⁺ were detected and spectral measurement at different time showed the transition between the two forms. Deprotonation of the radical was dependent on the concentration of water as proton acceptor. Later part of the thesis deals with efforts to covalently bind a catalytic unit to the previously characterized chromophore-electron relay module. The click chemistry approach was not successful to obtain the final photocatalytic assembly. Therefore bimolecular activation of a Mn salen catalyst was performed and formation of Mn(IV) species was observed. As a step towards utilization of these types of photocatalysts in a photoelectrochemical cell a [Ru(bpy)₃]²⁺ chromophore with phosphonate anchoring groups (Ru-Phosphonate) was synthesized and grafted on the surface of a TiO₂ mesoporous semiconductor surface anode to perform photocurrent measurements.

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.

This Ph.D. thesis addresses the structure-function relationship of the multimeric oxidoreductase WrbA with the principal aim being the explanation of the unusual kinetics of this enzyme in molecular terms, and thus getting an insight about its physiological role in bacteria. WrbA is a multimeric enzyme with FMN as a co-factor, catalyzing the oxidation of NADH by a two electrons transfer. Structure and function analysis of WrbA places this enzyme between bacterial flavodoxins and eukaryotic oxidoreductases in terms of its evolutionary relationship. The kinetic activity of WrbA was studied under varying conditions such as temperature, pH etc, and its kinetic mechanism was evaluated from parameters KM and Vmax and confirmed by product inhibition pattern experiments. Crystallization and proteolytic experiments also underpin the functional importance of the multimeric nature of WrbA and aid the understanding of the physiological role of this enzyme in molecular terms.

Neste estudo avaliamos o papel do metabolismo do triptofano (Trp) na homeostasia, na vaginose bacteriana e nas lesões cervicais associadas ao HPV. A importância do metabolismo do Trp se deve a sua ação na proliferação de microrganismos e de células do sistema imune. O consumo de triptofano tem sido identificado como uma forma de controlar o crescimento bacteriano limitando a infecção. Por outro lado, a oxidação de Trp produz quinurenina (QUIN), que tem papel chave na tolerância imunológica. A formação de QUIN se dá através das enzimas indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3- dioxigenase (TDO). A mais estudada delas no âmbito das infecções/ imuno escape é a enzima IDO. Mais recentemente, tem-se dado ênfase ao papel da TDO no câncer. Nesta dissertação, o interesse foi avaliar a expressão da IDO no epitélio cervicovaginal de mulheres com vaginose bacteriana e de IDO e TDO em amostras cervicais de mulheres com diferentes graus de lesão cervical associada ao HPV. Foram incluídas 165 mulheres atendidas no CAISM/UNICAMP, as quais foram divididas em dois grupos: grupo caso composto por mulheres com lesão de baixo ou alto grau e carcinoma invasor (n=42) e grupo controle composto por mulheres com citologia oncológica normal, independente de apresentar infecção genital (n=123). IDO foi avaliada por imunocitoquímica em citologia em base líquida e IDO e TDO em biópsias cervicais. Mulheres com vaginose bacteriana apresentaram expressão aumentada de IDO em células escamosas em comparação às mulheres sem vaginose bacteriana (OR=7.41; IC 95%= 2.50 a 21.4; p <0.0001). No epitélio vaginal normal com ou sem infecção por HPV houve uma expressão leve de IDO em células escamosas. Na presença de lesões ou carcinoma, houve um aumento no número de células escamosas displásicas e de leucócitos IDO-positivos; aumento de IDO também pôde ser observada em culturas de pele organotípicas transduzidas com as oncoproteínas E6/ E7 do HPV16. Nas lesões cervicais, assim como visto para a IDO, a TDO esteve expressa em leucócitos, especialmente os infiltrados na região estromal e na parede dos vasos sanguíneos. A expressão basal de IDO no epitélio cervical normal e sua regulação positiva na infecção por HPV e lesões associadas sugerem a participação do metabolismo do Trp nos mecanismos imunossupressores envolvidos na doença. Embora o papel do IDO já tenha sido abordada anteriormente, até onde sabemos esta é a primeira evidência da expressão de TDO no epitélio vaginal, na neoplasia intraepitelial cervical e carcinoma de células escamosas. Ainda, em leucócitos, especialmente aqueles com morfologia típica de polimorfonucleares, parecem ser importantes fontes de IDO na cérvix uterina. / In this study we evaluated the role of tryptophan (Trp) metabolism in cervix homeostasis, bacterial vaginosis and HPV-associated lesions. The importance of Trp metabolism is due to its action on microorganisms and immune cells. Tryptophan consumption has been identified as a way to controlling bacterial growth limiting infection. On the other hand, the oxidation of Trp produces kynurenine (Kyn) which plays a key role in immunological tolerance. The formation of Kyn occurs through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). IDO is the most studied of them within the context of infections / immune escape. More recently, TDO has also been considered in studies of cancer progression. In this thesis, we were interested in cervicovaginal epithelium IDO expression in women with bacterial vaginosis and of IDO and TDO in cervical samples of women with different degrees of cervical lesion associated with HPV. A total of 165 women attended at CAISM/UNICAMP were divided into two groups: a case group composed of women with low or high grade lesions and invasive carcinoma (n = 42) and a control group composed of women with normal cytology, independent to present genital infection (n =123). IDO was evaluated by immunocytochemistry in liquid-based cytology and IDO and TDO in cervical biopsies. Women with bacterial vaginosis had increased IDO expression in squamous cells compared to women without bacterial vaginosis (OR = 7.41, 95% CI = 2.50- 21.74; p<0.0001). In normal vaginal epithelium with or without HPV infection there was a mild IDO expression in squamous cells. In the presence of cervical intraepithelial lesions or squamous cell carcinoma, there was an increase in the number of IDO-positive dysplastic squamous cells and leukocytes; increase in IDO can also be observed in organotypic skin cultures transduced with HPV-16 E6/E7 oncoproteins. In cervical lesions, as observed for IDO, TDO was expressed in leukocytes, especially infiltrates in the stromal region and in the wall of blood vessels. The basal expression of IDO in the normal cervical epithelium and its positive regulation in HPV infection and associated lesions suggests the participation of Trp metabolism in the immunosuppressive mechanisms involved in the disease. Although some previous data have already considered the role of IDO, as far as we know this is the first evidence of the participation of TDO in the vaginal epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma. In addition, in leukocytes, especially those with a typical polymorphonuclear morphology, appear to be important sources of IDO in the uterine cervix.

Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides.

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.