O presente trabalho tem como objetivo geral a consolidação dos esforços iniciados anteriormente em nosso grupo para a utilização de marcação de spin sítio dirigida aliada à técnica de ressonância magnética eletrônica pulsada, em particular, ressonância dupla elétron-elétron (DEER), para medidas de distâncias entre sondas inseridas em moléculas biológicas. Como objetivo específico, interessa-nos a obtenção de informações estruturais de dois peptídeos pertencentes ao domínio de fusão da glicoproteína Spike do coronavírus causador da Síndrome Respiratória Aguda Grave (SARS) quando de sua ligação em modelos de membranas biológicas. Valemos-nos de uma abordagem conjunta envolvendo técnicas espectroscópicas, calorimétricas e computacionais para monitorarmos mudanças conformacionais nos peptídeos, suas conformações mais representativas e seus efeitos sobre a estrutura das membranas modelo. Os experimentos de calorimetria mostraram que os peptídeos perturbam fortemente o comportamento termotrópico de vesículas constituídas por fosfolipídios zwiteriônicos e negativamente carregados, sendo o efeito mais significativo na presença de lipídios negativos. Não somente a carga, mas também a estrutura da cabeça polar dos lipídios parece ter contribuição importante para a energética da interação. Os experimentos de dicroísmo circular mostraram que os peptídeos possuem alta flexibilidade conformacional, adotando diferentes estruturas secundárias em ambientes diversos. Uma mistura de conformações do SARSFP coexiste em solução aquosa e nos modelos de membranas, sugerindo alta plasticidade estrutural. Este peptídeo possui, ainda, alta capacidade de auto-associação e forma estruturas β e/ou agregados β regulares. Já o peptídeo SARSIFP adquire estrutura predominantemente α-helicoidal em micelas, estruturas β em lipossomos e conformações irregulares em água. Em particular, este peptídeo parece se ligar às membranas na forma de α-hélices, mas adquirir estruturas β em alta concentração. A flexibilidade conformacional dos peptídeos também foi estudada por dinâmica molecular (DM). Hélices, estruturas β, voltas, dobras e estruturas irregulares são visitadas durante as trajetórias dos dois peptídeos, mas o SARSIFP apresenta menor flexibilidade estrutural. O perfil de energia livre apresentado consiste de uma superfície plana, larga e rasa, sem grandes barreiras energéticas separando os diferentes estados conformacionais. A energia térmica à 300 K é suficiente para visitar boa parte do espaço conformacional acessível aos peptídeos ao longo dos parâmetros de ordem escolhidos. Por fim, obtivemos informações estruturais de análogos paramagnéticos dos peptídeos duplamente marcados com radicais nitróxidos quando em diferentes solventes e miméticos de membranas, além de estudarmos a oligomerização de derivados unicamente marcados. As distribuições de distâncias recuperadas dos sinais de DEER mostram que o SARSIFP adota primariamente α-hélices na presença dos miméticos, com provável formação de estruturas β na presença de micelas negativas. Para o SARSFP, uma larga distribuição de distâncias foi encontrada em todos os miméticos de membranas, refletindo a coexistência de conformações provavelmente bem compactas. Análogos unicamente marcados dos peptídeos ainda revelaram alta capacidade de formação de oligômeros em SDS-d25. Os resultados obtidos com esse conjunto de técnicas permitiram avanços consideráveis sobre as conformações adotadas pelos peptídeos nas diversas situações, o que pode revelar informações importantes acerca dos passos iniciais do mecanismo de fusão com membranas da glicoproteína Spike do coronavírus causador da SARS. / This thesis has the general goal of consolidating in our group the use of site directed spin labeling along with pulsed electron spin resonance, in particular double electron-electron resonance (DEER), for distance measurements in biological molecules. Our specific goal is to obtain structural information on two peptides belonging to the fusion domain of the spike glycoprotein from the SARS coronavirus when in the presence of membrane model systems. We used a joint approach involving spectroscopic, calorimetric, and computational techniques to monitor conformational changes in the peptides, their most representative conformations and their effects on the structure of model membranes. Calorimetric results showed that the peptides strongly perturb the thermotropic behavior of zwitterionic and negatively-charged lipid vesicles, with the largest effects seen with the later. Not only the charge, but also the lipid headgroup structure seems to be relevant for the energetics of the interaction. Circular dichroism experiments showed that the peptides present high conformational flexibility, assuming different secondary structures in diverse environments. A mixture of conformation of SARSFP coexists in aqueous solutions and in the membrane models, suggesting large structural plasticity. This peptide also showed high auto-association tendency forming β structures and/or regular β aggregates. On the other hand, the SARSIFP peptide is predominantly an α-helix when in micelles, β structures in lipossomes, and assumes irregular conformations in water. In particular, this peptide seems to bind to membranes as an α-helix, transitioning to β structures in high concentrations. The conformational flexibility of the peptides was also studied by molecular dynamics (MD). Helices, β structures, turns, hairpins and irregular structures are visited during the trajectories of both peptides, but SARSIFP presents less structural flexibility. The free energy profile is consistent with that of a plane, broad and shallow surface, without large energetic barriers separating the different conformational states. The thermal energy at 300 K is sufficient to make the peptides visit most of the conformational space accessible for a certain choice of order parameters. Lastly, we obtained structural information from paramagnetic analogs of the peptides, which were doubly-labeled with nitroxide radicals, in different solvents and membrane mimetics. We also studied the oligomerization process of singly-labeled analogs. The distance distributions determined from the DEER traces showed that SARSIFP adopts a primarily α-helix conformation in the presence of the mimetics, with the formation of β structures in the presence of negative micelles. DEER results for SARSFP showed a broad distribution of distances in all membrane mimetics, thus reflecting the coexistence of compact conformations. Singly-labeled analogs revealed the high tendency of formation of oligomers in SDS-d25. Our results allowed us to make considerable progress in understanding the conformations of the peptides in the conditions under investigation, which contributed with relevant information on the early steps of the membrane fusion mechanism carried out by the spike glycoprotein from the SARS coronavirus.

O presente trabalho tem como objetivo geral a consolidação dos esforços iniciados anteriormente em nosso grupo para a utilização de marcação de spin sítio dirigida aliada à técnica de ressonância magnética eletrônica pulsada, em particular, ressonância dupla elétron-elétron (DEER), para medidas de distâncias entre sondas inseridas em moléculas biológicas. Como objetivo específico, interessa-nos a obtenção de informações estruturais de dois peptídeos pertencentes ao domínio de fusão da glicoproteína Spike do coronavírus causador da Síndrome Respiratória Aguda Grave (SARS) quando de sua ligação em modelos de membranas biológicas. Valemos-nos de uma abordagem conjunta envolvendo técnicas espectroscópicas, calorimétricas e computacionais para monitorarmos mudanças conformacionais nos peptídeos, suas conformações mais representativas e seus efeitos sobre a estrutura das membranas modelo. Os experimentos de calorimetria mostraram que os peptídeos perturbam fortemente o comportamento termotrópico de vesículas constituídas por fosfolipídios zwiteriônicos e negativamente carregados, sendo o efeito mais significativo na presença de lipídios negativos. Não somente a carga, mas também a estrutura da cabeça polar dos lipídios parece ter contribuição importante para a energética da interação. Os experimentos de dicroísmo circular mostraram que os peptídeos possuem alta flexibilidade conformacional, adotando diferentes estruturas secundárias em ambientes diversos. Uma mistura de conformações do SARSFP coexiste em solução aquosa e nos modelos de membranas, sugerindo alta plasticidade estrutural. Este peptídeo possui, ainda, alta capacidade de auto-associação e forma estruturas β e/ou agregados β regulares. Já o peptídeo SARSIFP adquire estrutura predominantemente α-helicoidal em micelas, estruturas β em lipossomos e conformações irregulares em água. Em particular, este peptídeo parece se ligar às membranas na forma de α-hélices, mas adquirir estruturas β em alta concentração. A flexibilidade conformacional dos peptídeos também foi estudada por dinâmica molecular (DM). Hélices, estruturas β, voltas, dobras e estruturas irregulares são visitadas durante as trajetórias dos dois peptídeos, mas o SARSIFP apresenta menor flexibilidade estrutural. O perfil de energia livre apresentado consiste de uma superfície plana, larga e rasa, sem grandes barreiras energéticas separando os diferentes estados conformacionais. A energia térmica à 300 K é suficiente para visitar boa parte do espaço conformacional acessível aos peptídeos ao longo dos parâmetros de ordem escolhidos. Por fim, obtivemos informações estruturais de análogos paramagnéticos dos peptídeos duplamente marcados com radicais nitróxidos quando em diferentes solventes e miméticos de membranas, além de estudarmos a oligomerização de derivados unicamente marcados. As distribuições de distâncias recuperadas dos sinais de DEER mostram que o SARSIFP adota primariamente α-hélices na presença dos miméticos, com provável formação de estruturas β na presença de micelas negativas. Para o SARSFP, uma larga distribuição de distâncias foi encontrada em todos os miméticos de membranas, refletindo a coexistência de conformações provavelmente bem compactas. Análogos unicamente marcados dos peptídeos ainda revelaram alta capacidade de formação de oligômeros em SDS-d25. Os resultados obtidos com esse conjunto de técnicas permitiram avanços consideráveis sobre as conformações adotadas pelos peptídeos nas diversas situações, o que pode revelar informações importantes acerca dos passos iniciais do mecanismo de fusão com membranas da glicoproteína Spike do coronavírus causador da SARS. / This thesis has the general goal of consolidating in our group the use of site directed spin labeling along with pulsed electron spin resonance, in particular double electron-electron resonance (DEER), for distance measurements in biological molecules. Our specific goal is to obtain structural information on two peptides belonging to the fusion domain of the spike glycoprotein from the SARS coronavirus when in the presence of membrane model systems. We used a joint approach involving spectroscopic, calorimetric, and computational techniques to monitor conformational changes in the peptides, their most representative conformations and their effects on the structure of model membranes. Calorimetric results showed that the peptides strongly perturb the thermotropic behavior of zwitterionic and negatively-charged lipid vesicles, with the largest effects seen with the later. Not only the charge, but also the lipid headgroup structure seems to be relevant for the energetics of the interaction. Circular dichroism experiments showed that the peptides present high conformational flexibility, assuming different secondary structures in diverse environments. A mixture of conformation of SARSFP coexists in aqueous solutions and in the membrane models, suggesting large structural plasticity. This peptide also showed high auto-association tendency forming β structures and/or regular β aggregates. On the other hand, the SARSIFP peptide is predominantly an α-helix when in micelles, β structures in lipossomes, and assumes irregular conformations in water. In particular, this peptide seems to bind to membranes as an α-helix, transitioning to β structures in high concentrations. The conformational flexibility of the peptides was also studied by molecular dynamics (MD). Helices, β structures, turns, hairpins and irregular structures are visited during the trajectories of both peptides, but SARSIFP presents less structural flexibility. The free energy profile is consistent with that of a plane, broad and shallow surface, without large energetic barriers separating the different conformational states. The thermal energy at 300 K is sufficient to make the peptides visit most of the conformational space accessible for a certain choice of order parameters. Lastly, we obtained structural information from paramagnetic analogs of the peptides, which were doubly-labeled with nitroxide radicals, in different solvents and membrane mimetics. We also studied the oligomerization process of singly-labeled analogs. The distance distributions determined from the DEER traces showed that SARSIFP adopts a primarily α-helix conformation in the presence of the mimetics, with the formation of β structures in the presence of negative micelles. DEER results for SARSFP showed a broad distribution of distances in all membrane mimetics, thus reflecting the coexistence of compact conformations. Singly-labeled analogs revealed the high tendency of formation of oligomers in SDS-d25. Our results allowed us to make considerable progress in understanding the conformations of the peptides in the conditions under investigation, which contributed with relevant information on the early steps of the membrane fusion mechanism carried out by the spike glycoprotein from the SARS coronavirus.

<p>This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.</p><p>The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.</p><p>mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.</p><p>Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.</p>

Le virus de l’hépatite C (VHC) est, encore aujourd’hui, un problème de santé mondiale majeur entraînant dans certains cas des cirrhoses et des hépatocarcinomes. De nombreux efforts ont été fournis depuis les années 80 afin de développer un traitement efficace et sûr de cette infection touchant les hépatocytes. Le traitement interféron/ribavirine, utilisé dans les années 2000, a aujourd’hui été remplacé par des thérapies utilisant des agents antiviraux directs, beaucoup plus efficaces. Ces traitements restent cependant perfectibles notamment du fait de certains effets secondaires, de leur coût élevé et de potentielles interactions médicamenteuses avec d’autres composés thérapeutiques. L’équipe de glycochimie du Laboratoire de Chimie Biologique s’est intéressée à la synthèse de C-nucléosides analogues de la ribavirine. Parmi-eux, un composé, le SRO91, s’est révélé efficace contre des réplicons du VHC et présente une faible toxicité. Dans le but d’améliorer l’activité anti-VHC du SRO91, deux axes ont été développés dans ce projet : l’adressage vers les cellules du foie, et l’amélioration de la pénétration cellulaire. Un premier conjugué entre un galactoside et SRO91 a ainsi été synthétisé, afin de profiter de la forte interaction du galactose avec les récepteurs aux asialoglycoprotéines, principalement exprimés à la surface des hépatocytes. Afin d’améliorer sa pénétration cellulaire, le nucléoside a également été conjugué à des peptides de fusion du VHC. Ces séquences peptidiques très hydrophobes sont capables de s’insérer dans la membrane cellulaire et de permettre la fusion. Trois peptides ont été sélectionnés en se basant sur la littérature : HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) et HCV7 (SWHINRTALNSNDS), synthétisés par SPPS puis conjugués au nucléoside ou à un fluorophore. L’activité membranotropique des peptides sur des liposomes a alors été étudiée par calorimétrie (DSC et ITC), spectrofluorescence et microscopie à épifluorescence. Ces études ont ainsi permis de montrer que, parmi les séquences sélectionnées, HCV7 semble montrer la meilleure activité en pénétration membranaire alors que HCV6 s’est révélé être la séquence la plus fusogénique. / Hepatitis C virus (HCV) is a global healthcare issue responsible for cirrhosis and hepatocarcinoma. Strong efforts have been made since the 80’s to develop efficient and safe treatments for this liver infection. Hence, the treatment based on interferon/ribavirin, developed in 2002, has been replaced by much more efficient therapies involving direct-acting antivirals. However, the different side-effects, high cost and possible drug-drug interactions make room for improvements to this treatment. In the Laboratoire de Chimie Biologique, several C-nucleosides, analogs of ribavirin have been developed. Among them, one compound, named SRO91, seems effective against HCV replicons with low toxicity. This thesis work focused on improving SRO91 anti-HCV activity by implementing a targeting strategy and enhancing cell-penetration. We built our targeting strategy on the strong interaction between galactose and asialoglycoprotein receptors. Thus, a SRO91-galactose conjugate was synthesized, in order to address the antiviral to hepatocytes. To enhance cell-penetration we conjugated our nucleoside to HCV fusion peptides, since these highly hydrophobic sequences are able to anchor in cell membranes, leading to fusion. Three peptides were selected based on the literature: HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) and HCV7 (SWHINRTALNSNDS), synthesized by SPPS and conjugated to SRO91 or a fluorescent tag. Several techniques were used to study the membranotropic activity of theses sequences on liposomes as membrane models, including calorimetry (DSC and ITC), spectrofluorescence and epifluorescence microscopy. Thus, among the selected peptides, HCV7 seems to be the more potent as a membrane-penetrating agent but HCV6 shows the best fusogenic activity.

This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR. The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes. mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system. Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.