La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium. / The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.

La Enhanced Cyan Fluorescent Protein (ECFP) est un variant spectral de la Green Fluorescent Protein extraite de la méduse Aequaria Victoria (GFPav). La ECFP, émettant dans le cyan, est un des donneurs les plus utilisés dans les études de transfert résonnant d’énergie d’excitation et est integré dans de nombreuses constructions de biosenseurs. Pourtant, elle souffre de nombreux inconvenients. Notamment elle pésente des propriétés photophysiques complexes et une forte sensibilité environnementale qui sont des freins à une interprétation quantitative de ses signaux de fluorescence en imagerie cellulaire. Notre objectif vise à mettre au point, grâce à l’introduction d’un minimum de mutations, un dérivé de la ECFP présentant des propriétés d’émission simplifiées et performantes ainsi qu’une faible sensibilité environementale. Des mutations ont été introduites, par mutagenèse dirigée, à deux positions clefs de la séquence peptidique de la ECFP ce qui a permis de générer des dérivés présentant des propriétés photophysiques et une sensibilité au pH modulées. En particulier, nous avons réussit à générer une protéine fluorescente, l’Aquamarine, aux propriétés photophysiques quasi-idéales caractérisée par un rendement quantique de l’ordre de 0,9 et des déclins d'émission de fluorescence quasi-monexponentiels. Elle présente également une sensibilité au pH fortement réduite avec un pH de demi-transition acide de 3,3.Ce manuscrit présente l’étude détaillée des propriétés d’émission de fluorescence des diverses protéines générées. Plusieurs paramètres présentant un intérêt particulièrement important pour une utilisation adéquate en imagerie de fluorescence ont été évalués. Outre la sensibilité au pH établie sur une large gamme de pH (2,5-11), une attention particulière a été portée sur les performances photophysiques (monoexponentialité du déclin d'émission de fluorescence, durée de vie moyenne, rendement quantique, brillance,…) de ces dérivés. De plus, grâce à des expériences de dichroïsme circulaire, des informations sur les changements structuraux, dont ces dérivés sont le siège à pH très acide, ont été obtenues. Enfin, l’examen détaillé des données spectroscopiques stationnaires et résolues en temps a permis de mettre en lumière l’existence de plusieurs espèces émissives contribuant à la photophysique de ces protéines et à l’origine de leur transition acide. L’ensemble de ces résultats constitue une première approche pour une meilleure compréhension de la relation structure-photophysique-dynamique de la ECFP et de ces dérivés. / The Enhanced Cyan Fluorescent Protein (ECFP) is a variant of Green Fluorescent Protein extracted from the jellyfish Aequaria Victoria (GFPav). The ECFP, emitting in the cyan, is one of the most donors used in studies of resonant transfer excitation energy and is integrated in many constructions of biosensors. However, it suffers from several drawbacks. In particular, it presents a complex photophysical properties and high environmental sensitivity that are obstacles to its quantitative interpretation of fluorescence signals in cellular imaging. Our goal is to develop, through the introduction of minimum mutations, a derivative of the ECFP with simplified emission properties and low environmental sensitivity.Mutations were introduced by site-directed mutagenesis, into two positions in the peptide sequence of ECFP which helped to generate derivatives with modulated photophysical properties and low pH sensitivity. In particular, we have managed to generate a fluorescent protein, Aquamarine, with almost ideal photophysical properties characterized by a quantum yield of about 0.9 and pure single exponential fluorescence decay. It also has a greatly reduced sensitivity to the pH with half transition point near 3.3.This manuscript presents a detailed study of fluorescence properties of various proteins generated. Several parameters for proper use in fluorescence imaging were evaluated. In addition to the pH sensitivity based on a wide range of pH (2.5 to 11), particular attention was paid to the photophysical performance (simpler fluorescence emission decay, average lifetime, quantum yield, brightness, ...) of these derivatives. In addition, we studied structural changes on these derivatives at acidic pH by circular dichroism. Finally, a detailed examination of the steady state fluorescence and time-resolved fluorescence helped to highlight the existence of several emissive species contributing to the photophysics of these proteins and the origin of their acid transition. These results constitute a first approach to a better understanding of the structure-photophysical dynamics of ECFP-and these derivatives

Das aus dem Apoprotein Opsin und dem kovalent gebundenen Liganden bestehende Rhodopsin dient als Modellsystem für den Aktivierungsmechanismus der größten Klasse von G-Protein-gekoppelten Rezeptoren (GPCR). Infolge einer photochemischen Reaktion vollführt Rhodopsin eine Bewegungsabfolge von Sekundärstrukturelementen, wodurch es aktiviert wird, das G-Protein bindet und den Stimulus auf zellinterne Signalwege überträgt. Mithilfe der ortsspezifischen Mutagenese wurden Mutanten des bovinen Rhodopsins erzeugt, in eine künstliche Lipidumgebung eingelagert und hauptsächlich mittels FTIR-Spektroskopie untersucht. Anhand der Y191F- und Y192F-Mutanten konnte die Translokation des transienten Gegenions der Schiffschen Base Glu181 während der Aktivierung bestimmt werden. Die Interaktionen des Tyr206 sind für die gekoppelte Bewegung von EL2 und TM5 mitbestimmend, was mittels Y206F-Mutante gezeigt wurde. Eine Anhäufung von Methioninen auf der cytoplasmatischen Seite des Rezeptors ist u.a. für das Ausklappen der TM6 zuständig. Diese Bewegung ist wichtige Determinante der Rezeptoraktivierung. Hierfür wurden insgesamt fünf Mutanten verwendet. Im zweiten, hauptsächlichen Teil der Arbeit wird das bislang kaum untersuchte humane Rhodopsin mit dem bovinen Rezeptor verglichen. Ausgehend von verschiedenen Dunkelzuständen, konnte gezeigt werden, dass die Aktivierungsmechanismen beider Rezeptoren voneinander divergieren, um letztlich bei der Bildung der aktiven Spezies wieder zu konvergieren. Über die Analyse der Aminosäuresequenzen der Mammalia-Rhodopsine wurden zwei Bereiche hoher Variabilität identifiziert, die u.a. die molekulare Ursache für diese Diskrepanzen liefern. Diese Feststellung wurde mit human-bovinen-Rhodopsinchimären bewiesen. Ergänzend zu dieser Studie wurde Schafsrhodopsin einem Vergleich sowohl mit bovinem als auch mit humanem Rezeptor unterzogen. Es zeigte, als eine weitere natürlich vorkommende Variante des Lichtrezeptors, einen eigenständigen Weg der Aktivierung. / Rhodopsin, which consists of the apoprotein opsin and its covalently bound ligand, is used as a model system to understand the activation mechanism of the large family of G protein coupled receptors (GPCRs). As a result of a photochemical reaction, rhodopsin undergoes activating structural changes, enabling it to bind the G protein and transmitting the stimulus to intracellular signaling pathways. In the first part of this work, site-directed mutants of bovine rhodopsin were produced, incorporated into an artificial lipid environment, and studied mainly by FTIR spectroscopy. The translocation of the transient Schiff base counterion (Glu181) during the activation process was determined using the Y191F- and Y192F-mutants. The interactions of Tyr206 contributed to the coupled movement of EL2 and TM5, which was shown by Y206F-mutant. A striking accumulation of methionines on the cytoplasmic side of the receptor was observed to be a key-player for the activating outward motion of TM6. In the second and primary part of this work, human rhodopsin, which has been rarely studied, was compared with the bovine receptor. Starting from various dark states, it was shown that the activation mechanisms of both receptors diverge from each other and yet ultimately converge in the formation of the active species. By analyzing the amino acid sequences of mammalian rhodopsins, two regions of high variability were identified, which provide the molecular basis for these discrepancies. This finding was verified by the investigation of human/bovine rhodopsin chimeras. In addition to this study, ovine rhodopsin was compared with both the bovine and human forms. It showed, as another naturally occurring variant of the light receptor, an independent pathway of activation.

Flavonoids are secondary metabolites that are important in plant defense, protection and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. A flavonol-specific 3-O-GT enzyme has been identified and cloned from leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3-O-GTs from grape (Vitis vinifera) and grapefruit (Citrus paradisi) were modeled against F7-O-GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-O-GT that were identified through homology modeling. Analyses of the enzyme activity of the mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzyme inactive with flavonol substrates. Mutation of glycine 392 to glutamate (G392E) was active at 80% relative to the wild type. The mutant enzyme also did not show broadened acceptor specificity as it also favored flavonols as the preferred acceptor substrate. The glucosylation products of the active mutant enzyme will be analyzed to determine if this resulted in a change in regiospecificity.

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

La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium. / The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.

Οι μεταγραφικοί παράγοντες COUP-TF (Chicken Ovalbumin Upstream Promoter Transcription Factor) ανήκουν στην υπεροικογένεια των υποδοχέων στεροειδών/ θυρεοειδών ορμονών και θεωρούνται “ορφανοί”, αφού μέχρι στιγμής δεν έχει βρεθεί το υπεύθυνο πρόσδεμα για την ενεργοποίηση τους. Πειραματικές διαδικασίες στο εργαστήριο μας έχουν δείξει ότι στο πρωτογενές μετάγραφο των COUP-TFs συμβαίνει εναλλακτικό μάτισμα που έχει ως αποτέλεσμα την παραγωγή δύο mRNAs που κωδικοποιούν δύο πρωτεΐνες οι οποίες διαφέρουν ως προς το μέγεθος λόγω της εισαγωγής 21 επίπρόσθετων αμινοξέων στην καρβοξυτελική περιοχή (Carboxy Terminal Extension) της περιοχής πρόσδεσης στο DNA (DBD). Πειράματα EMSA με τη χρήση in vitro μεταφρασμένων πρωτεϊνών, αποκάλυψαν ότι η μεγάλη πρωτεΐνη δεν μπορεί να προσδεθεί σε κανένα COUP-TF στοιχείο απόκρισης. Επίσης παρατηρήθηκε ότι παρουσία της μεγάλης πρωτεΐνης, η ικανότητα της μικρής πρωτεΐνης να προσδένεται στο DNA μειώνεται με ανταγωνιστικό τρόπο, οδηγώντας στο συμπέρασμα ότι το ετεροδιμερές πρωτεϊνικό σύμπλοκο δεν μπορεί να προσδεθεί στο DNA. Σκοπός μας είναι να ερευνήσουμε το ρόλο της ένθεσης των 21 αμινοξέων στην μεγάλη πρωτεΐνη, ως προς την ικανότητα πρόσδεσης της στο DNA. Στον αχινό Paracentrotus lividus, η αμινοξική ένθεση στην καρβοξυτελική περιοχή (CTE) της μεγάλης πρωτεΐνης περιέχει δύο προλίνες. Η υπόθεση μας είναι ότι αυτές οι δύο προλίνες παίζουν σημαντικό ρόλο στην στερεοδιαμόρφωση της πρωτεΐνης, επηρεάζοντας την ικανότητα πρόσδεσης στο DNA. Για να ελέγξουμε την υπόθεση αυτή, προκαλέσαμε σημειακές μεταλλάξεις, μεταλλάσσοντας συγχρόνως τις δύο προλίνες σε αλανίνες αλλά κάθε μία προλίνη σε αλανίνη μεμονωμένα, καθώς επίσης μελετήσαμε και μια σειρά εσωτερικών αμινοξικών ελλειμάτων μέσα στην ένθεση των 21 αμινοξέων στην καρβοξυτελική περιοχή. Το σύνολο των μεταλλάξεων έδειξε ότι οι μεταλλαγμένες μεγάλες πρωτεΐνες δεν προσδένονται στο DNA καθώς επίσης ότι οι μεταλλαγμένες μεγάλες πρωτεΐνες ετεροδιμερίζονται πιο αποτελεσματικά με την μικρή ισομορφή πιθανότατα λόγω επαγόμενης αλλαγής της στερεοδιαμόρφωσης της μεταλλαγμένης πρωτεΐνης. Επίσης μελετήσαμε την εξάπλωση του εναλλακτικού ματίσματός στα Δευτεροστόμια, χρησιμοποιώντας ειδικούς εκφυλισμένους εκκινητές σε πειράματα PCR. Εναλλακτικό μάτισμα των μεταγράφων COUP-TF παρατηρήθηκε επίσης στους οργανισμούς Sphaerechinus granularis (Εχινόδερμο) και Saccoglossus kowalevskii (Ημιχορδωτό). / COUP-TFs (Chicken Ovalbumin Upstream Promoter- Transcription Factors) belong to the superfamily of steroid/thyroid hormone receptors and they are consider orphans since the proper ligand that activates them is not yet found. Experimental procedures in our laboratory have shown that in Echinoderms the alternative splicing of the COUP-TF primary transcript results in two mRNAs which encode two protein variants that differ by a 21 amino acid insertion in the Carboxy Terminal Extension (CTE) of the DNA Binding Domain (DBD). EMSA experiments with the use of in vitro translated proteins revealed that the large protein variant is incapable of binding any COUP-TF response elements. Furthermore, in the presence of the large variant, the small COUP-TF protein ability to bind DNA is diminished in an antagonistic way, suggesting that the heterodimeric protein is also incapable of DNA binding. Our aim is to investigate the role of the 21aa insertion in the large variant regarding the DNA binding affinity. In sea urchins the CTE insertion in the large variant contains two prolines. Our hypothesis is that these two prolines play an important role in the protein‟s conformation which in turn is responsible for the loss of DNA binding. To check this, we created point mutations by mutating both prolines to alanines simultaneously and then each proline to alanine separately. We also analyzed a series of internal amino acid deletions within the 21aa insertion of the CTE. All the mutations proved that the large mutated proteins are incapable of binding DNA and that they heterodimerize more effectively with the small protein variant possibly because of the changed conformation of the large protein variant. We also studied the alternative splicing among Deuterostomes, by using degenerate primers in PCR experiments. We observed that alternative splicing of COUP-TF transcripts occurs in the sea urchin Sphaerechinus granularis and in the hemichordate Saccoglossus kowalevskii.

Abstract NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13–14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function. Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G>A and m.3865A>G mutations and the ND6-affecting m.14498T>C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype. Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G>A and m.14498T>C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A>G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1. The results corroborate the pathogenicity of the m.14498T>C and m.3376G>A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme. / Tiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen.

The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis. DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested. AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity. The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance. Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.