KirBac channels are prokaryotic homologs of eukaryotic inwardly-rectifying potassium channels, which have served as models for gaining insight into the structure of eukaryotic channels. This thesis focuses on the structure-function relationship in these channels. The first part of this study concerns a novel KirBac channel, KirBac9.2, which contains a unique amino acid sequence in the place of the canonical GYG selectivity filter. Although expressed and purified in a stable and functional form, the protein did not form well-diffracting crystals. Functional studies suggest that KirBac9.2 is non-selective for monovalent cations and a random mutagenesis screen identified a number of activatory mutants in the cytoplasmic domains of the channel. A full electrophysiological investigation of KirBac9.2 channel function is beyond the scope of this study. However, initial studies suggest that it is possible to record currents from KirBac9.2 channels reconstituted into lipid bilayers. The second part of this thesis investigates KirBac3.1, which is a classical KirBac channel containing the consensus GYG sequence for potassium selectivity. Five high resolution structures of a mutant channel are reported, which suggest a new feature in the gating mechanism of KirBac3.1 where a rotation of the cytoplasmic domains is linked to a change in the electrostatic environment of the cytoplasmic cavity. In addition, a functional study of the KirBac3.1 showed that the channel is highly pH sensitive.

No description available.

Every cell is enclosed by a membrane which gives structure to the cell and allows for the passage of nutrients and wastes into and out of the cell. Membranes are made up of amphiphilic lipid molecules, with one water-soluble end, and one hydrophobic end. Naturally occurring and synthetic membranes are made up of double-chained amphiphiles derived from hydrocarbons. Recently, a novel class of amphiphilic molecules derived from fluorocarbons have been reported. The properties of fluorinated amphiphiles are very different to that of hydrocarbon based amphiphiles. Fluorinated amphiphiles have been previously reported to be useful in the studies of membrane proteins. In this thesis, we explore some novel uses of fluorinated amphiphiles. <b>Chapter one</b>: Provides a comprehensive review of the properties of fluorocarbon-based amphiphiles and discusses the existing uses of fluorinated amphiphiles in biochemical and biomedical research. <b>Chapter two</b>: Describes some of the important materials and methods used in this thesis including a detailed description of the proteins used and the working principles behind the techniques used in the study. <b>Chapter three</b>: Looks at the stability of pre-formed planar lipid bilayers in the presence of fluorinated amphiphiles (F-amphiphiles), and characterizes the behaviour of alpha-haemolysin and other proteins in liposomes and planar lipid bilayers in the presence of F-amphiphiles. We found that F-amphiphiles have an inhibitory effect on the insertion of protein into lipid bilayers, and this property has been exploited to control the number of proteins in the bilayer. <b>Chapter four</b>: Using droplet interface bilayers, we investigate the electrical properties and behaviour of protein(s) in bilayers formed by F-amphiphiles. The results obtained with fluorinated bilayers are compared with results obtained in conventional DPhPC lipid bilayers. This is the first ever report to carry out such an investigation and it provides insights into the formation, stability and utility of fluorinated bilayers. <b>Chapter five</b>: In Chapter five, we explore another aspect of droplet interface bilayers: the feasibility of using droplet interface bilayers to screen for membrane protein libraries. I have chosen to focus on certain fundamental aspects of the screening process that are sufficient to establish the feasibility of the method and to act as the proof of concept. <b>Chapter six</b>: Summarizes all the important results in the thesis and discusses some possible future directions of this project.

Bacterial chemotaxis is the movement of bacteria towards or away from chemical stimuli in the surrounding media. Bacteria respond to chemotactic signals through chemoreceptors which bind specific ligands and transduce signals through a modified two-component system. Typical chemoreceptors bind a ligand in the periplasm and signal across the inner membrane to the cytoplasmic chemosensory array through the inner membrane. Bacterial chemoreceptors must integrate multiple signals within an array of different receptor homologues to a single output. Chemoreceptors act cooperatively to allow a rapid signal spread across the array and large signal gain. Chemoreceptors adapt to a signal by chemical modification of their cytoplasmic domains in order respond across a wide range of effector concentrations. How bacterial chemoreceptors transduce signals through the inner membrane, integrate multiple effector responses, signal cooperatively and adapt to result in a single output signal is not currently fully known. In Rhodobacter sphaeroides, additional complexity arises from the presence of multiple homologues of various chemotactic components, notably the array scaffold protein CheW. Decoding this signalling mechanism and heterogeneity involved in this system is important in decoding the action of a biological system, with implications for biotechnology and synthetic biology. This study used the two model systems Escherichia coli and R. sphaeroides to analyse the mechanism of signalling through bacterial chemoreceptors. Rational design of activity-shifting chemoreceptor mutations was undertaken and these variants were analysed in phenotypic and fluorescence localisation studies. Molecular-dynamics simulations showed an increase in flexibility of chemoreceptors corresponds to a decrease in kinase output activity, which was determined by the computational tracking of bacteria free-swimming in media. Fluorescence recovery after photobleaching was used to show that this increase in flexibility results in a decrease in binding of receptors to their array scaffold proteins. A two-hybrid screen also suggested that inter-receptor affinity is also likely to decrease. These results show that signalling through chemoreceptors is likely through a mechanism involving the selective flexibility of chemoreceptor cytoplasmic domains. Analysis of R. sphaeroides chemoreceptors and CheW scaffold proteins in E. coli showed that it should be possible to design, from the bottom-up, a functional bacterial chemotaxis system in order to analyse individual protein specificity. Expression of R. sphaeroides MCPs in this E. coli system show the reconstitution of a chemotactic array, but not one capable of signalling specifically to proposed attractants. Results gained from this system suggest the R. sphaeroides CheW proteins are not homologous and their differential binding affinities may allow array activity 'fine-tuning'.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

The structures obtained from homology modeling methods are of intermediate resolution 1-3Ã… from true structure. Energy minimization methods allow us to refine the proteins and obtain native like structures. Previous work shows that some of these methods performed well on soluble proteins. So we extended this work on membrane proteins. Prediction of membrane protein structures is a particularly important, since they are important biological drug targets, and since their number is vanishingly small, as a result of the inherent difficulties in working with these molecules experimentally. Hence there is a pressing need for alternative computational protein structure prediction methods. This work tests the ability of common molecular mechanics potential functions (AMBER99/03) and a hybrid knowledge-based potential function (KB_0.1) to refine near-native structures of membrane proteins in vacuo. A web based utility for protein refinement has been developed and deployed based on the KB_0.1 potential to refine proteins.

Les synapses sont des structures fortement compartimentées remplies de complexes de protéines intéragissant entre elles. La fente synaptique reliant les compartiments pré- et post-synaptiques est une zone adhésive de 20 nm d'épaisseur, contenant des protéines d'adhésion et des récepteurs neurotransmetteurs. Les progrès dans l'imagerie à haute résolution offrent une vision améliorée de l'organisation dynamique des protéines synaptiques. Cependant, une évaluation quantitative de la concentration et du niveau d'oligomérisation de ces complexes reste difficile. Ceci est en partie dû au fait que les méthodes traditionnelles d'identification des récepteurs s'appuient sur des anticorps, dont la taille relativement grande (~ 15 nm) peut conduire à un empêchement stérique et un biais de localisation, alors que leur divalence provoque une réticulation des protéines. Ici, nous avons étudié l'impact de la valence de la sonde et des récepteurs sur la diffusion et l'organisation de 3 protéines synaptiques : Neurexin1β, neuroligine1 et la sous-unité du récepteur kainate GluK2. Nous avons utilisé une technique de single molecule pull down pour caractériser la composition de la sous-unité de ces protéines en observant des protéines possédant un tag GFP immobilisées sur un subtrat en utilisant une illumination TIRF. La neurexin1β présente essentiellement un step de photoblanchiment, tandis que la neuroligine1 présente principalement 2 steps, et GluK2 montre plusieurs steps, confirmant que ces protéines se rassemblent respectivement en monomères, dimères et tétramères. Ensuite, nous avons utilisé le FRAP pour surveiller la diffusion membranaire des 3 protéines marquées avec des sondes de valence différente. Nous avons étiqueté des protéines recombinantes portant un marqueur N-terminal biotinylé avec une streptavidine monovalente, divalente ou tétravalente (ou avec un anticorps anti-biotine), tous conjugués aux mêmes fluorophore organiques. Nous avons également utilisé une technique de STORM pour caractériser le niveau d'agrégation des 3 protéines en réponse aux différentes sondes. Nous montrons des effets drastiques en fonction de la nature de la protéine utilisée et de la valence de la sonde, suggérant que la diffusion est ralentie par l'agrégation des récepteurs, mettant ainsi en évidence les enjeux cruciaux dans les stratégies d'étiquetage, en particulier dans des espaces confinés tels que les synapses / Synapses are highly compartmentalized structures packed with interacting protein complexes. The synaptic cleft bridging pre- and post-synaptic compartments is an adhesive zone ~20 nm thick, containing adhesion proteins and neurotransmitter receptors. Progress in super-resolution imaging offers an improved view of the dynamical organization of synaptic proteins. However, a quantitative assessment of the concentration and oligomerization level of those complexes remains difficult. This is in part because traditional ways to label receptors rely on antibodies, whose relatively large size (~15 nm) may lead to steric hindrance and localization bias, while their divalence causes protein cross-linking. Here, we studied the impact of probe and receptor valence on the diffusion and organization of 3 synaptic proteins: neurexin1β, neuroligin1, and the GluK2 kainate receptor subunit. We used single molecule pull-down to characterize the subunit composition of those proteins, by observing immobilized GFP-tagged proteins under TIRF illumination. Neurexin1β shows essentially 1 photo-bleaching step, while neuroligin1 exhibits mostly 2 steps, and GluK2 shows multiple steps, confirming that those proteins assemble into monomers, dimers, and tetramers, respectively. Then, we used FRAP to monitor the surface diffusion of the 3 proteins labeled with probes of different valence. We labeled recombinant proteins carrying a biotinylated N-terminal tag with monomeric, dimeric, or tetrameric streptavidin (or with biotin antibody), all conjugated to the same organic dyes. We also used STORM to characterize the aggregation level of the 3 proteins in response to the different probes. We show drastic effects depending on the nature of the protein used and the probe valence, suggesting that diffusion is slowed down by receptor aggregation, thereby highlighting the crucial issues at stake in labelling strategies, especially in confined spaces such as synapses.

Na,K-ATPase é uma proteína de membrana que tem como função manter o equilíbrio osmótico nas células pela hidrólise de ATP. A ouabaína (OUA) se liga a Na,K-ATPase e é capaz de ativar cascatas de sinalização. As subunidades a da Na,K-ATPase possuem 4 isoformas que são distribuídas de forma diferenciada nos tecidos. As células da glia são importantes na resposta contra lesões no cérebro e também controlam a inflamação. Dados na literatura mostram que a OUA tem efeito protetor em alguns tipos de dano. O objetivo do estudo é avaliar a função da isoforma a2 na cultura de células da glia em resposta à OUA e ao LPS. Nós investigamos a ação da OUA em diversas concentrações e LPS (1g/mL) na viabilidade celular (LDH) e proliferação celular (MTT). O LPS foi utilizado como modelo de inflamação e uma das perguntas era se o tratamento prévio com OUA, seria capaz de reverter a ativação do fator de transcrição NF-kB que está envolvido com inflamação. O pré-tratamento com OUA diminuiu a ativação do NF-kB induzida pelo LPS. Após, nós silenciamos a isoforma a2 das células da glia com RNAi. Os nossos dados mostram que o pré-tratamento com OUA reverte o efeito na ativação do NF-kB causado pelo LPS. Provavelmente, a isoforma a2 está relacionada com alguma via de sinalização que interage com a via do LPS. / Na,K-ATPase is a conserved membrane protein which maintains the osmotic balance in the cell by the hydrolysis of ATP. Ouabain (OUA) binds to Na,K-ATPase and it can activate signaling pathways. The a subunits of Na,K-ATPase have 4 isoforms which are distributed in a different pattern in the tissues. Glial cells have an important role in the response against injury and they also control inflammation. Some data have reported that OUA can protect against some types of injury. The aim of this study is to evaluate the role of a2 isoform in glial cells in response to OUA and LPS stimulus. We investigated the action of OUA and LPS in cell viability (LDH) and cell proliferation (MTT). LPS was used as a model of inflammation and one of our questions was if the treatment with OUA before LPS was capable of reduce the activation of the transcription factor NF-kB which is involved in inflammation. The pre-treatment with OUA decreased the NF-kB activation induced by LPS. We also silenced the a2 isoform in culture glial cells with iRNA. Taken together our data showed that OUA pretreatment reversed the NF-kB activation induced by LPS in primary cultures of glial cells from mice. Probably,the a2 isoform is related with some signaling pathway that interacts with the LPS pathway.

Entre cepas diferentes de P. falciparum existe uma grande variação entre as sequências das famílias de genes variantes. Um motivo para esta grande variedade é o fato que a maioria dos genes variantes se encontra em regiões subteloméricas e que o parasita é capaz de recombinar telômeros heterólogos durante a meiose (recombinação ectópica), levando a uma nova distribuição e a criação de novos genes variantes. Além desse fenômeno que ocorre durante a fase sexual do parasita, foi considerado que recombinações também podem ocorrer durante a fase mitótica na fase assexuada sanguínea. Neste estudo, procuramos monitorar a importância desta recombinação ectópica na geração de novos genes var em amostras de campo da Amazônia brasileira. Em experimentos paralelos elucidamos se existe recombinação ectópica também durante divisões puramente mitóticas. Observamos que muitos genes var que são compartilhados entre isolados mudam raramente ou não mudam de posição cromossômica. Observamos que no caso de mudança de posição cromossômica muitas vezes ocorreu duplicação do lócus. Muitos dos genes var compartilhados se encontraram em cromossomos 5-6 e 9-7. Por monitoramento de clones de 3D7 após 180 gerações não observamos nenhuma translocação de genes var subtelomérico ou telomérico indicando que a recombinação ectópica em mitoses é de fato um evento raro. / Different strains of the causative agent of malaria, Plasmodium falciparum, possess greatly varying repertoires of variant antigen encoding gene families. One reason for this variety lies in the fact that most of the variant gene families are found in subtelomeric regions. The parasite is able to recombine heterologous telomers during meiosis through a process coined ectopic recombination, potentially leading to a new distribution and creation of variant genes. Due to morphological similarities of chromosome end clustering in sexual as well as in asexual forms, it was hypothesized that ectopic recombination may also occur in mitotic asexual blood stage parasites. Herein we monitor the occurrence of ectopic recombination in field samples from the Brazilian Amazon. In parallel, we elucidated whether ectopic recombination also takes place in purely mitotic divisions. We observed that many var genes which are shared among isolates rarely change their chromosomal position. When a change occurred, we often observed chromosomal locus duplication and many of the shared genes were found on chromosomes 5-9 or 5-10. After outgrowth of the 3D7 strain for 200 generations with intermittent cloning we did not observe any translocation of telomeric or subtelomeric var genes, indicating that ectopic recombination in mitosis is a rare event.

Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Helen Alves Penha / Banca: José Belasque Júnior / Banca: Daniel Guariz Pinheiro / A Xanthomonas citri subsp. citri (Xac) é a bactéria causadora da doença cancro cítrico. A doença atinge todas as variedades comerciais de citros e, o entendimento da relação fitopatogênica permitirá conhecer formas mais eficientes de controle da bactéria. Com o sequenciamento e análise do genoma e transcriptoma da Xac, foram identificadas ORFs que, provavelmente, estão envolvidas em processos de ataque e colonização da bactéria. Para iniciar a caracterização de uma destas ORFs foram utilizadas, neste estudo, as estratégias de mutação sítio-dirigida de PCR por sobreposição e extensão e modelagem molecular. A ORF XAC0239, é predita como proteína de membrana plasmática, e por homologia, possivelmente, pode fazer parte de uma proteína transportadora de zinco, além disso, há indícios de que é regulada por um fator sigma. A mutação teve efeito negativo sobre a ação de celulases e motilidade swimming e efeito positivo sobre a formação de biofilme e a multiplicação bacteriana in vivo. Aqui é sugerido que na falta desta proteína, houve pouca absorção de zinco, necessário para a formação de biofilme e consequentemente, para a patogenicidade. Além disso, sugestiona-se que, como subterfúgio, a bactéria superexpressou genes para produção e/ou ação de celulases, tentando atacar o hospedeiro de forma alternativa. / Xanthomonas citri subsp. citri (Xac) causes the citrus canker disease. The disease reaches all commercial varieties of citrus and, the understanding of the phytopathogenic relationship allow knowing the ways of control of bacteria. With sequencing of the Xac genome and the realization of a transcriptome, ORFs have been identified that are probably involved in bacterial attack and colonization processes. To initiate the characterization of one of these ORFs, in this study, the strategies of site-directed mutagenesis by overlap extension PCR and molecular modeling were used. The ORF XAC0239 is predicted as plasma membrane protein, and by homology may possibly be part of a zinc transporter protein, moreover, there are indications that it is regulated by a sigma factor. The mutation had a negative effect at the cellulases action and swimming motility and a positive effect on biofilm formation and in vivo multiplication. Here it is suggested that in the absence of this protein, there was little absorption of zinc, necessary for biofilm formation and consequently for pathogenicity. In addition, it is suggested that, as a subterfuge, the bacteria overexpressed genes for production and / or cellulases action, trying to attack the host on alternative way. / Mestre