The first section of this thesis concerns the study of interactions between the intense local plasmonic field generated by nanostructure and a well known photosynthetic protein system, bacteriorhodopsin (bR). bR is a membrane protein responsible for proton transport. Among the many intermediates formed upon photoexcitations, two of the most relevant have been studied. The intermediates under studies were I460 and M412, and their decay dynamics were measured in presence of the plasmonic field generated by the excitation of their surface electrons using visible photons. Both intermediates decay lifetime were affected when the plasmonic field was turned on, and it was verify that thermal effect were not the source of the change in dynamic. The second part concerns the investigation of third-order nonlinearity of a series of extended conjugated squaraine dyes in the telecommunication spectral region. Their nonlinearity is measured via Degenerate Four Wave Mixing and Z-scan as function of the dyes increasing conjugation length and number of squarylium groups. The dyes produced large real and imaginary values for the third order nonlinearity in the 1300-1500 nm range which makes them attractive for optical limiting type of applications.

The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

Monotopic proteins represent a specialized group of membrane proteins in that they are engaged in biochemical events taking place at the membrane interface. In particular, the monotopic lipid-synthesizing enzymes are able to synthesize amphiphilic lipid products by catalyzing two biochemically distinct molecules (substrates) at the membrane interface. Thus, from an evolutionary point of view, anchoring into the membrane interface enables monotopic enzymes to confer sensitivity to a changing environment by regulating their activities in the lipid biosynthetic pathways in order to maintain a certain membrane homeostasis. We are focused on a plant lipid-synthesizing enzyme DGD2 involved in phosphate shortage stress, and analyzed the potentially important lipid anchoring segments of it, by a set of biochemical and biophysical approaches. A mechanism was proposed to explain how DGD2 adjusts its activity to maintain a proper membrane. In addition, a multivariate-based bioinformatics approach was used to predict the lipid-binding segments for GT-B fold monotopic enzymes. In contrast, a soluble protein Myr1 from yeast, implicated in vesicular traffic, was also proposed to be a membrane stress sensor as it is able to exert different binding properties to stressed membranes, which is probably due to the presence of strongly plus-charged clusters in the protein. Moreover, a bacterial monotopic enzyme MGS was found to be able to induce massive amounts of intracellular vesicles in Escherichia coli cells. The mechanisms involve several steps: binding, bilayer lateral expansion, stimulation of lipid synthesis, and membrane bending. Proteolytic and mutant studies indicate that plus-charged residues and the scaffold-like structure of MGS are crucial for the vesiculation process. Hence, a number of features are involved governing the behaviour of monotopic membrane proteins at the lipid bilayer interface. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

Chlamydia trachomatis infections are among the most frequently reported causes of human sexually transmitted infection. In Australia, the reported rate of infection in 2004 reached 175 per 100,000 population, the highest rate since surveillance of the condition began in 1991. Severe adverse sequelae that commonly occur following progression of the infection from the lower to the upper genital tract include pelvic inflammatory disease, infertility and ectopic pregnancy. However the frequent prevalance of asymptomatic infection makes diagnosis and treatment often late and therefore ineffective against upper genital tract complications. Hence there is a great need to develop a vaccine to protect against the sexual transmission of C.trachomatis. Despite many years of research investigating potential vaccine strategies to prevent sexually transmitted C.trachomatis infections, there remains no commercially available C.trachomatis vaccine. Early research showed that the use of live, attenuated or inactivated whole Chlamydia as a vaccine was not a viable option due to adverse effects caused by immunopathogenic cellular components. The early human vaccine trials that utilized whole chlamydial cells and resulted in exacerbated disease when immunized individuals were re-exposed to Chlamydia have led to the investigation of chlamydial subunit components as potential vaccine antigens. The most widely investigated vaccine candidate antigen is the major outer membrane protein (MOMP) as it is known to be immunogenic and surface exposed. Much research using this antigen has been undertaken with the antigen being delivered as a protein, peptide or DNA, via many mucosal and systemic routes of immunization, and in combination with various vaccine adjuvants. However, at best only partial protection against a chlamydial genital tract infection has been achieved. Only a few alternative candidate antigens have been investigated as potential vaccine targets to protect against chlamydial infections. These include the outer membrane porin PorB, the large cysteine rich outer membrane protein Omp2 and the heat shock proteins DnaK and GroEL. Although other candidate antigens have been predicted in various models of chlamydial infection (Finco et al., 2005; Stemke-Hale et al., 2005; Li et al., 2006), few have been tested for their protective efficacy. The aim of this study was to use expression library immunization to screen the whole C.muridarum genome for novel vaccine candidates capable of protecting against a chlamydial genital tract infection. C.muridarum was selected as the disease model for chlamydial genital tract infection as it has similarities to C.trachomatis in pathogenesis, immune response to infection and gene content and order. Once protective antigens had been isolated from an expression library, these were screened individually for immunogenicity and protective efficacy in the C.muridarum model of infection. An expression library containing over 21,000 recombinant C.muridarum clones was constructed and divided into pools of clones. DNA was extracted from these pools and used to immunize mice through gene gun technology, delivering 1μg of DNA to the abdomen of mice. Following the immunization regime, mice were challenged intra-vaginally with live C.muridarum as this route of infection best resembles the natural route of infection that is responsible for the sexual transmission of C.trachomatis in humans. Four in vivo screens of the C.muridarum expression library, each time using reduced numbers of clones, resulted in the identification of seven novel vaccine antigens that conferred protection against a genital tract challenge infection in mice. These warrant further investigation as vaccine antigens in the development of a vaccine against C.trachomatis infection. The identified antigens include antigens not conventionally believed to be potential vaccine candidates such as hypothetical proteins and housekeeping genes, including a DNA gyrase subunit, TC0462, and the ATP-dependent Clp protease, ATP-binding subunit ClpC, TC0559. Other antigens identified were more traditional, surface exposed vaccine targets that have not been previously investigated as vaccine targets, including a novel outer membrane protein, TC0512, a polymorphic membrane protein, TC0693, and TC0850, a protein of the type three secretion system, a family of proteins that allow gram-negative bacteria to inject virulence related proteins into the cytoplasm of a host cell. All antigens were shown to be partially protective with the putative outer membrane protein TC0512 showing an overall reduction in chlamydial burden of 55% and other antigens showing overall reductions in chlamydial burden of 26 - 44%. These antigens were also either capable of stimulating an immune response, or predicted to contain epitopes that may stimulate strong immune responses and so warrant further investigation as vaccine antigens to protect against chlamydial genital tract infections. The results of this research demonstrate that it is possible to identify novel vaccine targets through screening an expression library in a disease model. This study has identified several novel vaccine targets that are partially-protective against a C.muridarum infection and that are thought to be capable of stimulating strong immune responses. These antigens have high homology with C.trachomatis sequences, indicating that they have potential as vaccine candidates capable of protecting against the serovars of C.trachomatis that cause sexually transmitted infections in humans. Although the protection observed in this study was only partial, the immunization strategy utilised only fragments of the genes, an immunization mechanism known to elicit Th2 type immune responses, and no adjuvant to enhance the immunogenicity of the antigens. Through different immunization routes and in conjunction with adjuvants that stimulate Th1 type immune responses, complete protection against chlamydial genital tract infections may be achieved.

Le transporteur NTT1 est responsable pour l'import d'ATP dans les chloroplastes afin de maintenir le métabolisme en période d'activité réduite ou nulle de la photosynthèse. Cependant, le mécanisme moléculaire de ce transporteur est encore méconnu, essentiellement du à la difficulté de manipulation des protéines membranaires. Nous avons réussi à développer un protocole pour la production de ce transporteur, permettant une bon rendement de solubilisation et obtention de protéines purifiées pour des études structurales. Combinant des caractérisations biochimiques et biophysiques, nous avons pu identifier des conditions de préparation d'échantillons qui ont mené aux premiers cristaux. Afin d'étendre notre connaissance sur les transporteurs de nucléotides, nous avons également entrepris des études structurales et fonctionnelles sur AAC, le transporteur ADP/ATP des mitochondries. AAC et NTT1 appartiennent à des familles de protéines différentes mais ont des fonctions voisines. À partir de la première structure d'AAC déjà connue, nous avons recherché par des criblages in silico de nouvelles molécules se liant au transporteur de façon compétitive avec le nucléotide et pouvant ainsi inhiber le transport. Les outils de docking ont été mis en place et ont permis à partir d'une librairie de 75000 composés d'identifier 17 molécules. Ensuite, nous avons testés ces molécules expérimentalement et montré qu'une d'entre elles inhibent le transport. De plus, trois nouveaux analogues d'ADP ont également été identifiés comme inhibiteurs. / Chloroplast NTT1 mediates external supply of ATP in the plastids, which is pre- requisite for the maintenance of plastid metabolism during limited or missing photosynthetic activity. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. We have successfully developed a protocol for the production of this transporter, that is compatible for efficient solubilization and good yield of purified protein. Combining biochemical and biophysical analyses, we could characterize the protein solution and identify conditions from which first crystals could be obtained. To further extend our work with nucleotide transporter, structural and functional studies on mitochondrial ADP/ATP carrier (AAC) have been performed. AAC and NTT1 belong to different families but exhibit similar functional features. To discover small-molecule inhibitors of AAC's transport for structural studies, virtual docking of compounds was performed into the AAC active site, using the already known structure of AAC. Docking tools were installed and screening a large library of 75,000 compounds allowed the identification of 17 molecules. The experimental binding assay for AAC, revealed that one of the compounds has inhibitory activity in the micromolar regime. In addition, 3 novel ADP analogues showing inhibitory effect against ATP transport of AAC have also been identified.

O sorotipo O125ac:H6 compreende amostras de Escherichia coli enteropatogênica atípicas que apresentam o padrão de adesão agregativa (AA) em células HEp-2. A construção de um banco de mutantes da amostra protótipo Ec292/84 com o transposon TnphoA gerou quatro mutantes não aderentes. O objetivo deste estudo foi a caracterização genotípica e fenotípica desses mutantes. As regiões adjacentes à inserção do TnphoA no mutante Ec2921/84::01 foram amplificadas, clonadas e seqüenciadas, revelando que a inserção do TnphoA ocorreu no gene secD, parte do sistema de secreção de proteínas do tipo 2 (SST2). O perfil de proteínas de membrana externa (OMP) dos mutantes, em comparação com a amostra selvagem, revelou a ausência de proteínas de 21 e 30 kDa nos mutantes. Um antissoro obtido contra o extrato de OMP da amostra protótipo inibiu o padrão AA e reconheceu a proteína de 30 kDa em immunoblottings com extratos de OMP. Esses dados indicam que esta proteína está envolvida no estabelecimento do padrão AA de E. coli O125ac:H6 e que essa proteína é transportada através do SST2. / The serotype O125ac:H6 comprises atypical Enteropathogenic Escherichia coli strains that express the aggregative adherence (AA) pattern to HEp-2 cells. We obtained four nonadherent mutants using TnphoA insertion in the Ec292/84 strain. The aim of this study was the genetic and phenotypic characterization of these mutants. The genetic analysis of the mutants revealed that the insertion of TnphoA ocurred in the secD gene, part of the bacterial type 2 secretion system (T2SS). The mutant outer membrane proteins (OMP) profile, in comparison to the prototype strain, demonstrated the lack of expression of proteins of 21 and 30 kDa in the mutant profile. An antiserum raised against the OMP extract of the prototype strain, in addition to inhibit the AA pattern, recognized the 30 kDa protein in immunoblotting assays with OMP extract. These data indicate this OMP is involved in the establishment of the AA pattern presented by the atypical EPEC strains of the O125ac:H6 serotype, and that this protein is transported via the T2SS.

Due to the inherent problems with their structural elucidation in the laboratory, the computational prediction of membrane protein structure is an essential step toward understanding the function of these leading targets for drug discovery. In this work, the development of a genetic algorithm technique is described that is able to generate predictive 3D structures of membrane proteins in an ab initio fashion that possess high stability and similarity to the native structure. This is accomplished through optimisation of the distances between TM regions and the end-on rotation of each TM helix. The starting point for the genetic algorithm is from the model of general TM region arrangement predicted using the TMRelate program. From these approximate starting coordinates, the TMBuilder program is used to generate the helical backbone 3D coordinates. The amino acid side chains are constructed using the MaxSprout algorithm. The genetic algorithm is designed to represent a TM protein structure by encoding each alpha carbon atom starting position, the starting atom of the initial residue of each helix, and operates by manipulating these starting positions. To evaluate each predicted structure, the SwissPDBViewer software (incorporating the GROMOS force field software) is employed to calculate the free potential energy. For the first time, a GA has been successfully applied to the problem of predicting membrane protein structure. Comparison between newly predicted structures (tests) and the native structure (control) indicate that the developed GA approach represents an efficient and fast method for refinement of predicted TM protein structures. Further enhancement of the performance of the GA allows the TMGA system to generate predictive structures with comparable energetic stability and reasonable structural similarity to the native structure.

Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Les TSPO forment une famille ancienne et hautement conservée à travers l’évolution de protéines à 5 domaines transmembranaires, que l’on retrouve aussi bien chez les animaux que les plantes, ou encore les bactéries. La TSPO animale (ou TSPO1), la plus étudiée des TSPO à ce jour, se trouve majoritairement dans les tissus stéroïdiens où sa fonction précise est controversée. Chez certaines espèces animales, il existe une isoforme, la TSPO2, localisée dans la membrane plasmique des globules rouges alors que la TSPO1 est mitochondriale. La fonction de la TSPO2 n’est pas bien caractérisée. La TSPO végétale, localisée dans le réticulum endoplasmique, possède une extension N-terminale que n’ont pas les TSPO animale et bactérienne. Elle semble être impliquée dans la régulation du stress, tout comme la TSPO bactérienne. Les études structure/fonction des différentes TSPO réalisées dans ce travail ont nécessité leur production par voie recombinante car elles sont naturellement peu abondantes.Nous avons produit la TSPO1 murine marquée 15N,13C par surexpression dans la bactérie E. coli. Puis la protéine purifiée en détergent (SDS et DPC) a été étudiée par différentes techniques (CD, fluorescence, RMN). L’ajout du ligand spécifique de la TSPO1, le PK11195, stabilise une conformation en DPC ce qui a permis en 2014 la résolution de sa structure, par RMN du liquide, par une équipe allemande. Afin d’étudier la TSPO1 dans un environnement plus proche de sa membrane native, nous l’avons reconstituée dans des liposomes DMPC/DMPE et étudiée par RMN du solide. Les 1ers résultats sont encourageants et ouvrent une nouvelle approche expérimentale pour la détermination de sa structure en présence et, plus particulièrement, en absence de ligand. La surexpression de la TSPO2 humaine dans la bactérie E. coli s’est avérée difficile et nous avons dû la réalisée par système acellulaire (cell-free). Les quantités obtenues par cette méthode permettent d’envisager le développement futur des études des relations structure/fonction.La production et la purification du Nter de la TSPO d’A. thaliana marqué 15N,13C ont permis la détermination de sa structure par RMN du liquide. Son interaction avec des lipides chargés mise en évidence par les études RMN, suggère une nouvelle fonction de l’AtTSPO dans le trafic lipidique. / TSPO are five-transmembrane domain proteins that form a protein family highly conserved throughout evolution and that are found in animals as well as in plants and bacteria.Animal TSPO (referred to as TSPO1), the most studied TSPO, is highly expressed in tissues involved in steroid biosynthesis where its precise role remains controversial. In some animal species the presence of a less characterized TSPO isoform, TSPO2, has been reported. TSPO2 was found to be located in the plasma membrane of red blood cells whereas TSPO1 is located in mitochondrial outer membrane. Plant TSPO, which is located in the endoplasmic reticulum, possesses an N-terminal extension that is absent in bacterial and animal TSPO. This TSPO, along with the bacterial TSPO, seems to be involved in stress regulation.The structural and functional studies of TSPO proteins conducted in this work required their production through recombinant expression because they are naturally non-abundant proteins.We made use of E. coli to produce the recombinant 15N,13C-labelled mouse TSPO1. Then the protein purified in detergent was studied through several methods (CD, fluorescence, NMR). High-affinity binding of PK11195 to TSPO1 stabilizes a conformation in DPC, which made possible the structure determination of the protein in solution by NMR by a German team. We have incorporated TSPO1 into DMPC/DPME liposomes in order to provide a native-like environment and we then studied it by solid-state NMR. Preliminary results are encouraging and open up a new approach for TSPO1 structure determination in presence or in absence of ligand.Human TSPO2 overexpression in E. coli proved to be difficult and we therefore use the cell-free method. The amounts we obtained by this method allows us to consider future developments of structurefunction relationship studies.Production and purification of 13C,15N labelled N-terminal of A. thaliana TSPO have made it possible to determine its structure by liquid state NMR. Interaction of this peptide with charged lipids revealed by NMR, suggests a new fonction of AtTSPO in lipid trafficking.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.