This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.

This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling. / text

In this thesis, a 4.7Å crystal structure of the human mitochondria DNA polymerase γ catalytic complex is reported. Though the DNA substrate-binding site is not identifiable in the structure, two conformational changes in the enzyme architecture are described: 1) rotation of the distal monomer of the accessory subunit towards the catalytic subunit, and 2) shift of the thumb motif of the polymerase domain towards the active site. Both conformational changes suggest a structure of Pol γ in the DNA-bound state and in its active site “closed” conformation. / text

No description available.

Banana is the most important fruit crop in the world but ironically one of the crops least studied. This fruit constitutes a major staple food for millions of people in developing countries and also it is considered the highest selling fruit in the world market making this crop a very important export commodity for the producing countries. At the present time, one of the most significant constraints of banana production that causes significant economical losses are fungal diseases. Among these, Panama disease, also known as Fusarium wilt has been the most catastrophic. Panama disease is caused by the soil-borne fungus Fusarium oxysporum formae specialis (f.sp) cubense (FOC), which infects susceptible bananas through the roots causing a lethal vascular wilt. To date, the race 4 of this pathogen represents the most serious threat to banana production worldwide since most of the commercial cultivars are highly susceptible to this pathogen. Introduction of FOC resistance into commercial cultivars by conventional breeding has been difficult because edible bananas are sterile polyploids without seeds. Genetic transformation of banana, which has already been established in various laboratories around the world has the potential to solve this problem by transferring a FOC race 4 resistance gene into susceptible banana cultivars (eg. Cavendish cultivars). However, a FOC resistant (R) gene has not been isolated. Genes that confer resistance to Fusarium oxysporum have been isolated from tomato and melon using a map-based positional cloning approach. The tomato I2 and melon Fom-2 genes belong to the non-Toll/interleukin like receptors (TIR) subclass of nucleotide-binding site and leucine-rich repeat (NBS-LRR) R genes. These genes confer resistance only to certain races of F. oxysporum in their corresponding plant families limiting their use in other plant families. The fact that these two Fusarium resistance genes share the same basic non-TIR-NBS-LRR structure suggests a similar Fusarium resistance mechanism is shared between the families Solanaceae and Cucurbitaceae. This observation opens the possibility to find similar Fusarium resistance genes in other plant families including the Musaceae. A remarkable discovery of a population of the wild banana Musa acuminata subspecies (ssp.) malaccensis segregating for FOC race 4 resistance was made by Dr. Ivan Buddenhagen (University of California, Davis) in Southeast Asia. Research carried out at Queensland Department of Primary Industries (Australia) using this plant material has demonstrated that a single dominant gene is involved in FOC race 4 resistance (Dr. Mike Smith, unpublished results). Tissue-culture plantlets of this FOC race 4 segregating population were kindly provided to the Plant Biotechnology Program (Queensland University of Technology) by Dr. Mike Smith to be used in our research. This population holds the potential to assist in the isolation of a FOC race 4 resistance gene and other potential Fusarium resistance genes. The overall aims of this research were to isolate and characterise resistance gene candidates of the NBS-type from M. acuminata ssp. malaccensis and to identify and characterise potential Fusarium resistance genes using a combination of bioinformatics and gene expression analysis. Chapter 4 describes the isolation by degenerate PCR of five different classes of NBS sequences from banana (Musa acuminata ssp malaccensis) designated as resistance gene candidates (RGCs). Deduced amino acid sequences of the RGCs revealed the typical motifs present in the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses showed that the banana RGCs are related to non-TIR subclass of NBS sequences. The copy number of each class was estimated by Southern hybridisation and each RGC was found to be in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to Fusarium oxysporum f. sp. cubense (FOC) race 4. Four classes showed a constitutive expression profile whereas no expression was detected for one class in either tissue. Interestingly, a transcriptional polymorphism was found for RGC2 whose expression correlated with resistance to FOC race 4 suggesting a possible role of this gene in resistance to this devastating FOC race. Moreover, RGC2 along with RGC5 showed significant sequence similarity to the Fusarium resistance gene I2 from tomato and were chosen for further characterisation. The NBS sequences isolated in this study represent a valuable source of information that could be used to assist the cloning of functional R genes in banana. Chapter 5 describes the isolation and characterisation of the full open reading frame (ORF) of RGC2 and RGC5 cDNAs. The ORFs of these two banana RGCs were predicted to encode proteins that showed the typical structure of non-TIR-NBS-LRR resistance proteins. Homology searches using the entire ORF of RGC2 and RGC5 revealed significant sequence similarity to the Fusarium resistance gene I2 from tomato. Interestingly, the phylogenetic analysis showed that RGC2 and RGC5 were grouped within the same phylogenetic clade, along with the Fusarium resistance genes l2 and Fom-2. These findings suggest that the banana RGC2 and RGC5 are potential resistance gene candidates that could be associated with Fusarium resistance. The case of RGC2 is more remarkable because its expression was correlated to FOC race 4 resistance (Chapter 4). As a first step to test whether RGC2 has a role in FOC race 4 resistance, different expression constructs were made with the ORF of this sequence. One of the constructs contains a RGC2 putative promoter region that was successfully cloned in this work. These constructs will be used to transform susceptible banana plants that can then be challenged with FOC race 4 to assess whether resistance has been acquired by genetic complementation. The results of this thesis provide interesting insights about the structure, expression and phylogeny of two potential Fusarium resistance genes in banana, and provide a rational starting point for their functional characterisation. The information generated in this thesis may lead to the identification of a Fusarium resistance gene in banana in further studies and may also assist the cloning of Fusarium resistance genes in other plant species.

An increasing number of proteins with unknown function have their three-dimensional structure solved at high resolution. This situation, largely due to structural genomics initiatives, has been stimulating the development of automated structure-based function prediction methods. Knowledge of residues important for function – and more particularly – for binding can help automated prediction of function in different ways. The properties of a binding site such as its shape or amino acid composition can provide clues on the ligand that may bind to it. Also, having information on functionally important regions in similar proteins can refine the process of annotation transfer between homologues.<p>Experimental results indicate that functional residues often have an unfavourable contribution to the stability of the folded state of a protein. This observation is the underlying principle of several computational methods for predicting the location of functional sites in protein structures. These methods search protein structures for destabilising residues, with the assumption that these are likely to be important for function.<p>We have developed a method to detect clusters of destabilising residues which are in close spatial proximity within a protein structure. Individual residue contributions to protein stability are evaluated using detailed atomic models and an energy function based on fundamental physico-chemical principles.<p>Our overall aim in this work was to evaluate the overlap between these clusters of destabilising residues and known binding sites in proteins.<p>Unfortunately, reliable benchmark datasets of known binding sites in proteins are sorely lacking. Therefore, we have undertaken a comprehensive approach to define binding sites unambiguously from structural data. We have rigorously identified seven issues which should be considered when constructing datasets of binding sites to validate prediction methods, and we present the construction of two new datasets in which these problems are handled. In this regard, our work constitute a major improvement over previous studies in the field.<p>Our first dataset consists of 70 proteins with binding sites for diverse types of ligands (e.g. nucleic acids, metal ions) and was constructed using all available data, including literature curation. The second dataset contains 192 proteins with binding sites for small ligands and polysaccharides, does not require literature curation, and can therefore be automatically updated.<p>We have used our dataset of 70 proteins to evaluate the overlap between destabilising regions and binding sites (the second dataset of 192 proteins was not used for that evaluation as it constitutes a later improvement). The overlap is on average limited but significantly larger than random. The extent of the overlap varies with the type of bound ligand. Significant overlap is obtained for most polysaccharide- and small ligand-binding sites, whereas no overlap is observed for nucleic acid-binding sites. These differences are rationalised in terms of the geometry and energetics of the binding sites.<p>Although destabilising regions, as detected in this work, can in general not be used to predict all types of binding sites in protein structures, they can provide useful information, particularly on the location of binding sites for polysaccharides and small ligands.<p>In addition, our datasets of binding sites in proteins should help other researchers to derive and validate new function prediction methods. We also hope that the criteria which we use to define binding sites may be useful in setting future standards in other analyses. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La comparaison de site peut-elle vérifier l’hypothèse: «Les origines biosynthétiques des produits naturels leurs confèrent des activités biologiques»? Pour répondre à cette question, nous avons développé un outil modélisant les propriétés accessibles au solvant des sites de liaison. La méthode a montré des aspects intéressants, mais elle souffre d’une sensibilité aux coordonnées atomiques. Cependant, des méthodes existantes nous ont permis de prouver que l’hypothèse est valide pour la famille des flavonoïdes. Afin d’étendre l’étude, nous avons développé un procédé automatique capable de rechercher des structures d’enzymes de biosynthèse de produits naturels disposant de sites actifs capables de lier une molécule de petite taille. Nous avons trouvé les structures de 117 enzymes.Les structures nous ont permis de caractériser divers modes de liaison substrat-enzyme, nous indiquant l’empreinte biologique des produits naturels ne correspond pas toujours au modèle « clé- serrure ». / Can computational binding site similarity tools verify the hypothesis: “Biosynthetic moldings give potent biological activities to natural products”? To answer this question, we designed a tool modeling binding site properties according to solvent exposure. The method showed interesting characteristics but suffers from sensitivity to atomic coordinates. However, existing methods have delivered evidence that the hypothesis was valid for the flavonoid chemical class. In order to extend the study, we designed an automated pipeline capable of searching natural products biosynthetic enzyme structures embedding ligandable catalytic sites. We collected structures of 117 biosynthetic enzymes. Finally, according to structural investigations of biosynthetic enzymes, we characterized diverse substrate-enzyme binding-modes, suggesting that natural product biological imprints usually do not agree with the “key-lock” model.

Le présent travail porte sur l’étude de deux protéines de la famille des oxydases à hème-fer par des techniques de spectroscopie et d’électrochimie. Le premier chapitre décrit l’étude du cytochrome bo3 oxydase issue d’E. coli. Nous nous sommes intéressés à l’étude des interactions enzyme-quinone par l’utilisation de quinones avec des longueurs chaines isoprenyl différentes. Notre but est de mieux comprendre le rôle de la longueur de la chaine des quinones sur l’activité catalytique de l’enzyme et sur les propriétés redox des cofacteurs à hème. Dans l’étape suivante, on a étudié les résidus impliqués dans le site de liaison des quinones (haute affinité, QH). Plusieurs mutations de ces résidus sont étudiées pour mieux comprendre l’importance de chacun des résidus dans cette liaison. Dans la dernière partie de ce premier chapitre, la spectroscopie SEIRAS «spectroscopie d’absorption infrarouge exaltée de surface» est introduite comme une technique alternative pour l’étude des protéines membranaires. Dans le second chapitre, la protéine membranaire cNOR issue de P. denitrificans est étudiée. Nous nous sommes focalisés sur l’effet de différents environnements (pH, présence de protéo-liposomes) sur la stabilité de la cNOR. Pour ce faire, trois valeurs de pH (6.5, 7.5 et 8.5) sont choisies et quelques échantillons de cNOR sont reconstitués dans des protéo-liposomes. Enfin, le donneur de proton terminal (au centre binucléaire) dans la protéine cNOR était étudié. De plus, nous avons étudié les ligands des ions Ca2+ puisqu’il est proposé que le donneur de proton est situé proche de cette région. / This thesis is focused on the study of two members of the heme-copper oxidase family by using spectroscopic and electrochemical techniques. In the first chapter cytochrome bo3 oxidase from E. coli was studied. We focused on the quinone-enzyme interactions by using quinones with different isoprenyl chains. Our aim was to better understand the role of isoprenyl chain on the catalytic activity of the enzyme and the redox properties of the heme cofactors. In the next step we studied the residues that are suggested to be in the high-affinity (QH) quinone binding site. Several site-directed mutants of these residues were investigated in order to better understand the position of QH binding site and the importance of each residue. In the last part of this chapter surface-enhanced infrared absorption spectroscopy (SEIRAS) was introduced as an alternative technique to study the membrane proteins. In the second chapter cytochrome c dependent nitric oxide reducates (cNOR) from P. denitrificans was studied. We focused on the effect of different environment (pH, proteoliposomes) on the stability of cNOR. For that purpose three pH values (6.5, 7.5 and 8.5) was selected and some of the cNOR samples were reconstituted in liposomes. Finally, the terminal proton donor (to the binuclear center) in cNOR was investigated. We studied the ligands of the Ca2+ site in cNOR since it was suggested that the proton donor may be close to this area.