Tesi sul tema "Affinity labeling"

Unter den Techniken der chemischen Proteomik hat Capture Compound – Massenspektrometrie (CCMS) den Vorteil, Interaktionen von Molekülen mit geringer Affinität zueinander effektiv untersuchen zu können. CCMS beruht auf kleinen molekularen Sonden (Capture Compounds, CCs), die aus drei funktionalen Bestandteilen bestehen: die Selektivitätsfunktion, ist ein kleines Molekül, das mit einem Zielprotein eine schwache Wechselwirkung eingeht. Die zweite Funktionalität erlaubt kovalente Anhaftung der molekularen Sonde an Proteine. Der dritte Anteil erlaubt Detektion mit sehr guten Sensitivität; allerdings ist die Quantifizierung weiterhin ein Schwachpunkt dieser Technik. Ziel dieses Projektes ist, eine in CCMS verwendbare Quantifizierungsmethode zu entwickeln. Heutzutage gibt es zahlreiche MS-basierte Quantifizierungsstrategien; unsere beruht auf der Einführung von Lanthanoid-haltigen Labels – Metal Coded Affinity Tagging (MeCAT). In dieser Arbeit wurde erstmalig die erfolgreiche Verwendung mit Metall- Markern chemoproteomischer Sonden (CCs) zur Detektion und absoluten Quantifizierung von Zielproteinen mit schwacher Wechselwirkung etabliert. Mit den Experimenten an isolierten Enzymen und an lebenden Zellen wurde nachgewiesen, dass Metall-Marker keinen negativen Einfluss auf andere funktionelle Teile chemoproteomischer Sonden haben. CCs, die mit Lanthanoid-Chelaten funktionalisiert sind, zeigen ähnliche Affinität zu ihren Zielproteinen wie die Referenz-Sonden. Zudem erlauben Metall-Marker, die für diese Art molekularer Sonden verwendet werden, die Entwicklung einer element-basierten Technik zur Bilderzeugung. Der herausragende Vorteil der Metall-funktionalisierten CCs kombiniert mit ICP-MS ist, dass diese eine absolute Quantifizierung der Ausbeute der Quervernetzungen ermöglichen.
Capture compound mass spectrometry (CCMS) is a chemical proteomics technique that has the advantage of addressing low abundant target proteins in lysates as well as in living cells. The CCMS is based on small molecule probes (capture compounds) that consist of three functionalities: a small molecule (quite often it is a drug), which interacts with the target protein; the moiety that allows covalent attachment of the molecular probe to the protein; the one that allows detection. The detection moiety utilized for CCMS can offer high sensitivity; however, the challenge of absolute quantification is still a bottleneck of this technique. Metal Coded Affinity Tagging (MeCAT) is a quantitative approach based on the chemical labeling with lanthanide; it allows obtaining both the structural and quantitative information. In this work for the first time the successful utilization of chemoproteomic probes functionalized with a metal tag for the detection and absolute quantification of target proteins was established. With the experiments both on isolated enzymes and living cells it was determined that MeCAT does not negatively influence other functional parts of the probes; therefore, capture compounds functionalized with lanthanide chelates demonstrate similar affinity to the target as the reference probes. Moreover, metal tags utilized for this type of molecular probes can offer a promising elemental imaging technique. However, to achieve the sufficient resolution multiple metal tags per molecular probe are needed. The striking advantage of the approach of utilization metal functionalized capture compound combined with ICP-MS detection is that it allows absolute quantification of crosslink yield, what cannot be performed with other detection methods applied for this technology.

We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule.

QC 20140929

There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The

KCN-1 is known to reduce tumor growth 6-fold in mice implanted with LN229 glioma cells. Although this inhibitor is effective, the mechanism of action for KCN-1 is not well understood. Based on preliminary studies, KCN-1 reduces tumor growth by disrupting the HIF 1 (hypoxia-induced factor-1) pathway. The binding target of KCN-1 needs to be investigated in order to develop KCN-1 or its analogs for therapeutic applications. In this research, a molecule was designed and synthesized for the identification of the binding target of KCN-1. Specifically, this molecule contains the inhibitor (KCN-1), a photocleavable linker, beads, and the affinity label (L DOPA). When UV light shines on the linker, the trans-alkene isomerizes to cis-alkene and undergoes intramolecular ring-closing reaction, which helps cleave the immobilized bead from the linker. The immobilized bead is used to separate the binding fragment attached to the photocleavable linker from the solution after enzyme digestion. The affinity label (L-DOPA) reacts with a nucleophile from the binding target and creates a covalent bond. If the design is successful, this method is able to analyze the mass of the peptide sequence and determine the binding target of KCN-1.

Copper is an essential trace element and required for various biological processes, but free copper is toxic. Therefore, copper is tightly regulated in living cells and disruptions in this homeostatic machinery are implicated in numerous diseases. The current understanding of copper homeostasis is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level. Intracellular copper is primarily present in the monovalent oxidation state. Therefore, copper(I) selective fluorescent probes can be utilized for imaging exchangeable copper ions in live cells, but these probes are often lipophilic and hence poorly water soluble. To address this problem, water-soluble fluorescent probes with greatly improved contrast ratio and fluorescence quantum yield are characterized in this work. This work also describes a novel application of water-soluble fluorescent probes, in-gel detection of copper proteins with solvent accessible Cu(I) sites under non-denaturing conditions. Knowledge of copper(I) stability constants of proteins is important to elucidate the mechanisms of cellular copper homeostasis. Due to the high affinity of most Cu(I)-binding proteins, the stability constants cannot be determined directly by titration of the apo-protein with Cu(I). Therefore, accurate determination of Cu(I) stability constants of proteins critically depends on the Cu(I) affinity standards. However, the previously reported binding affinity values of the frequently used Cu(I) affinity standards are largely inconsistent impeding reliable data acquisition for the Cu(I) stability constants of proteins. To solve this problem, a set of water-soluble ligands are developed in this work that form colorless, air-stable copper(I)-complexes with 1:1 stoichiometry. These ligands can be applied as copper(I) buffering agents and affinity standards in order to study copper biochemistry. Copper(I) binding proteins are an integral part of the copper homeostatic machinery and they work in conjunction to regulate copper uptake, distribution, and excretion. However, available evidence indicates the existence of putative copper-binding proteins that are yet to be characterized. Therefore, several proteomics-based methods are developed in this work by employing the strategy to label Cu(I)-binding cysteines in a copper-dependent manner which lays the foundation for the identification of new copper proteins from cellular extracts.

Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogène
Shiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs

Les Métallo Protéases Matricielles (MMP) en tant qu'endopeptidases à zinc ont une large gamme de fonctions biologiques allant du remodelage tissulaire à la modulation de la réponse cellulaire. Une modification de leur activité protéolytique est souvent associée à de nombreux désordres biologiques. In vivo, ces protéases sont soumises à de nombreuses modifications post-traductionnelles. Elles sont sécrétées sous formes latentes à l'extérieur des cellules pour être ensuite transformées en forme fonctionnelles. Ces dernières sont ensuite inhibées par des inhibiteurs endogènes. En raison de leur sécrétion dans l’espace extra cellulaire, les MMP sous formes actives ont longtemps été considérées comme de simples ciseaux moléculaires capable de dégrader uniquement la matrice extracellulaire. Cependant, le remodelage tissulaire ne constitue pas la fonction unique et encore moins la fonction principale de ces enzymes. Elles peuvent en effet cliver une grande variété de substrats non matriciels et à ce titre sont impliquées dans la progression tumorale, l'immunité et l'inflammation. Pour ajouter une complexité supplémentaire à la biologie des MMP, il a été récemment montré que certaines MMP ont une localisation intracellulaire associée à des fonctions non protéolytiques. Ces observations, mais aussi celles montrant que ces protease participent à la progression de la maladie alors que d'autres ont une fonction protectrice, soulignent la nécessité de mieux documenter leur activation spatiale et temporelle dans divers contextes biologiques.Le profilage protéique basé sur l'activité vise à analyser l'état fonctionnel des protéines dans des échantillons biologiques complexes. À cette fin, des sondes basées sur l'activité (ABP), qui réagissent avec les enzymes en s’appuyant sur leur mécanisme catalytique, ont été développées pour la détection d’enzymes sous formes actives, notamment dans le cas des protéases à sérine et à cystéine. Une sonde basée sur l’activité (ABP) est classiquement composée : i) d’un groupement réactif conduisant à la modification covalente de résidus au sein du site actif de l’enzyme, ii) d’un motif de liaison imposant la sélectivité au groupement réactif et iii) d’un groupement rapporteur permettant la détection des enzymes ciblées. Cette approche ne s’applique toutefois pas aux MMP, pour lesquelles il n’existe pas de résidus nucléophiles conservés au sein du site actif. À cet égard, tous les ABP ciblant les MMP comportent un groupement photo activable qui, sous irradiation UV, favorise la formation du complexe covalent. De telles sondes photo sensibles ont permis de détecter les MMP sous leurs formes actives dans des tissus et des fluides, mais pas chez les animaux vivants au sein desquels l’étape de photo-activation ne peut être réalisé.Dans ce contexte, en nous appuyant sur un contexte structural favorable et en exploitant la chimie de l'acyl imidazole (LDAI) dirigée par un ligand, nous avons identifié une nouvelle série de sondes capables de modifier de manière covalente les MMP sans recourir à la photo-activation. Nous avons ainsi validé la capacité de ces sondes à marquer de manière sélective et efficace la MMP12 humaine in vitro et dans des protéomes complexes. Dans ce dernier cas, jusqu’à 50ng de hMMP12 correspondant à 0,05% du protéome total peuvent être détectés. Nous avons également déterminé l'identité de l’unique résidu modifié de façon covalente au sein du site actif de la hMMP-12 et vérifié que cette modification avait peu d'impact sur l’activité protéolytique de cette dernière. Nous avons démontré que cette approche permettait de détecter des MMP endogènes. Enfin, nous avons étendu cette stratégie de marquage à un panel plus large de MMP.En développant la première stratégie de marquage des formes actives de MMP «sans photo-activation», il semble maintenant possible d’envisager la détection de ces enzymes à la fois dans les protéomes complexes et in vivo
Matrix MetalloProteases (MMPs) as zinc endopeptidases have a wide range of biological functions, and changes in their proteolytic activity underlie many biological disorders. Since their proteolytic activity has to be tightly controlled to prevent tissue destruction, theses proteases are subjected to numerous posttranslational modifications in vivo. They are secreted under latent forms outside of the cells, and are subsequently processed into their functional form that can be further inhibited by endogenous inhibitors. Due to their delineated area of activation, MMP active forms have long been considered for their unique ability to degrade extracellular substrates. However, turnover and breakdown of the extracellular matrix are neither the sole nor the main function of MMPs. These enzymes can indeed process a wide variety of non-matrix substrates and are involved in the regulation of multiple aspects of tumor progression, immunity and inflammation. To add further complexity to MMPs biology, some members within the family were recently reported to have intracellular localization associated to non-proteolytic functions. These observations but also those evidencing that some MMPs participate in disease progression while others have a protective function, stress the need to better document their spatial and temporal activation in various biological contexts.Activity-based protein profiling (ABPP) aims to analyze the functional state of proteins within complex biological samples. To this purpose, activity-based probes (ABPs) that react with enzymes in a mechanism-based manner have been successfully developed for the profiling of several enzymes including serine and cysteine proteases. A typical Activity-Based probe (ABP) is composed of i) a reactive warhead, which reacts in a covalent manner with enzyme active site residues, ii) a targeting moiety that imposes selectivity upon the reactive group and iii) a detectable group for subsequent analyses. This approach is not applicable to MMPs, which lack a targetable nucleophile involved in the catalysis. In this respect, all ABPs directed to MMPs are affinity-based probes (AfBPs) containing within their structure a photo cross-linking group that promotes the formation of a covalent complex upon UV-irradiation. Such photoactivatable probes have been successfully developed for the detection of MMPs under their active forms in fluids and tissue extracts, but not in living animals where the photo-activation step is not feasible.By relying on a favorable structural context and by exploiting the ligand-directed acyl imidazole (LDAI) chemistry, we have identified a novel series of AfBPs capable of covalently modifying matrix metalloproteases without making use of photo-activation. These active-site-directed probes whose structure was derived from that of a MMP12 selective inhibitor harbored a reactive acyl imidazole in their P3' position. They demonstrated their labelling specificity in vitro by covalently modifying a single Lysine residue within the MMP-12 S3' region. We also showed that these probes only targeted functional states of hMMP-12 and spared forms whose active site was occluded either by a synthetic or a natural inhibitor. We have validated the ability of these chemical probes to efficiently label human MMP12 in complex proteomes. In this case, down to 50 ng of hMMP12 corresponding to 0.05% of the whole proteome can be labelled and detected by in-gel fluorescence analysis. We demonstrated that this approach also allowed detecting endogenous MMPs secreted by stimulated-macrophages. In addition, by modifying the nature of the targeting moiety, we have extended this affinity-labeling approach to six other MMPs.By developing the first “photo activation-free” strategy to covalently modify active forms of MMPs, the unresolved proteomic profiling of native MMPs should be now accessible both in complex proteomes and in preclinical model in which MMPs are potential relevant targets

Les objectifs de mes travaux de thèse étaient de développer de nouvelles méthodes d’identification, de caractérisation et de quantification de protéines, mieux adaptées à la diversité des études en protéomique, ce dont la biologie a besoin aujourd’hui. L’analyse protéomique par spectrométrie de masse est apparue comme un outil précieux et pertinent pour évaluer la qualité de l’isolement d’un complexe spécifique, et pour guider les biologistes dans les choix de la stratégie à adopter. La stratégie de marquage de N-terminomique développée a permis de caractériser un processus de maturation biologique en déterminant précisément les sites d’activation de la protéine Perséphone par marquage spécifique des extrémités N-terminales. Ce travail a permis d’élucider un nouveau mécanisme fin de régulation dans l’immunité innée chez la drosophile. De nouveaux modes de marquages ont été mis au point et les familles chimiques des réactifs de marquage étudiés permettront d’adapter au mieux les études de quantifications protéomiques à la nature et aux contraintes des études biologiques à mener
The aim of this work was to develop new methods for the identification, characterization and quantification of proteins best suited to a large diversity of proteomics studies, which is nowadays essential to biology. Our work has shown that proteomic analysis based on mass spectrometry can be a valuable and relevant tool to evaluate the isolation strategy efficiency set up for a specific complex and thus guide the biologists in their choice. The N-terminomic labeling strategy developed allowed us to describe a biological maturation process by determining precisely the Persephone protein activation sites using specific labeling of the successively generated N-terminal extremities. This work allowed elucidating a new regulation mechanism in the Drosophila innate immunity system. New chemical labeling reagents to target specific amino acids (cysteine, tyrosine and tryptophan) have been set up for fast mass-spectrometry based proteomics. These labeling strategies combined with proteomic tools will allow developing a robust and quantitative approach essential for biological studies

L'utilisation de l'acide para-chloromercuribenzene sulfonique (pcmbs) (reactif peu permeant des groupements thiols) comme marqueur differentiel du transporteur de saccharose des tissus foliaires de feve (vicio faba) revele une forte inhibition selective de l'absorption du saccharose. Cette inhibition est due au blocage du transporteur et non a celui de la pompe a protons fournissant l'energie necessaire au transport. Le point d'impact du pcmbs se situe au niveau du site actif de l'enzyme. Ce marquage differentiel a permis de mettre au point un test de reconnaissance des substrats par le transporteur de saccharose. Les resultats obtenus precisent les exigences stereochimiques du transporteur de saccharose et sont utilisables pour la synthese bioprogrammee de pesticides a systemie phloemienne

Etude des problèmes liés à l'utilisation des déshydrogénases a NAD(P) en synthèse organique. Etude, à l'aide d'analogues structuraux du NAD(P), de la 6-phosphogluconate déshydrogénase de Candida Utilis et de l'oestradiol déshydrogénase de placenta humain. Synthese d'un complexe covalent NAD-alcool déshydrogénase du foie de cheval, catalytiquement actif. Purification de la carnitine déshydrogénase de Pseudomonas Putida et la mise en oeuvre pour la synthèse stéréospécifique de la l-carnitine. Mise au point d'une électrode à enzyme spécifique de la l-carnitine

Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric regulation to the mouse enzyme and other eukaryotic reductases. The overall activity of the enzyme, which has two subunits (Rl and R2), is regulated through binding to ATP, which activates the enzyme, and dATP which seryes as an inhibitor. Both nucleotides bind to the same allosteric site, called the activity site, on the large subunit of RR. The specificity of the enzyme towards the four ribonucleoside diphosphate substrates is regulated by the binding of ATP, dATP, dTTP and GTp. Each of these nucleotides affects the reduction of a specific nucleoside diphosphate. Although this enzyme's allosteric regulation is kinetically well understood it has not been possible so far to gain further structural information about the location of the activity site and specificity site. The use of deletion mutants and photoaffinity labeling of the large subunit to identify the location of the binding sites is the incentive for this thesis. With the introduction of 6xHis/Nickel Nitrilo-tri-acetic acid (Ni-NTA) chromatography, the purification of the large subunit was improved in the E. coli and vaccinia virus/T7 RNA polymerase hybrid system. The purification of several deletion mutant forms of the large subunit was also attempted, but it was not possible to purify any of them from either of the expression systems. The purified full-length large subunit obtained with the Ni-NTA-chromatography system was used for a photoaffinity labeling experiment with [³²P]dATP and [³²P]dTTP. The labeled proteins were proteolytically digested to find out about the specificity of the labeling experiment and also to map the binding site of the nucleotide. It was found that labeling of dATP yielded few discrete bands indicating specific binding, while a comparable experiment with dTTP indicated less specific binding, based on a larger number of labeled bands. In competition experiments with non-radioactive nucleotides, vaccinia virus R1 featured the same properties as the mouse and E. coli counterparts. This is consistent with data from kinetic experiments, which also establish the same kinetic properties between vaccinia virus RR with those of mouse and E. coli (RR). To identify the sequence of the fragments carrying the label the digests were subjected to mass spectrometric analysis. However, it was not possible to determine the sequence of the labeled fragment by mass spectrometry due to poor spectral resolution.
Graduation date: 1994

Indiana University-Purdue University Indianapolis (IUPUI)
The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

Il existe un lien étroit entre la structure tridimensionnelle et la fonction cellulaire de l’ARN. Il est donc essentiel d’effectuer des études structurales de molécules d’ARN telles que les riborégulateurs afin de mieux caractériser leurs mécanismes d’action. Une technique de choix, permettant d’obtenir de l’information structurale sur les molécules d’ARN est la spectroscopie RMN. Cette technique est toutefois limitée par deux difficultés majeures. Premièrement, la préparation d’une quantité d’ARN nécessaire à ce type d’étude est un processus long et ardu. Afin de résoudre ce problème, notre laboratoire a développé une technique rapide de purification des ARN par affinité, utilisant une étiquette ARiBo. La deuxième difficulté provient du grand recouvrement des signaux présents sur les spectres RMN de molécules d’ARN. Ce recouvrement est proportionnel à la taille de la molécule étudiée, rendant la détermination de structures d’ARN de plus de 15 kDa extrêmement complexe. La solution émergeante à ce problème est le marquage isotopique spécifique des ARN. Cependant, les protocoles élaborées jusqu’à maintenant sont très coûteux, requièrent plusieurs semaines de manipulation en laboratoire et procurent de faibles rendements. Ce mémoire présente une nouvelle stratégie de marquage isotopique spécifique d’ARN fonctionnels basée sur la purification par affinité ARiBo. Cette approche comprend la séparation et la purification de nucléotides marqués, une ligation enzymatique sur support solide, ainsi que la purification d’ARN par affinité sans restriction de séquence. La nouvelle stratégie développée permet un marquage isotopique rapide et efficace d’ARN fonctionnels et devrait faciliter la détermination de structures d’ARN de grandes tailles par spectroscopie RMN.
The tridimensional structure of a given RNA molecule is closely linked to its cellular function. For this reason, it is crucial to study the structure of RNA molecules, such as riboswitches, to characterize their mechanism of action. To do so, NMR spectroscopy is often used to gather structural data on RNA molecules in solution. However, this approach is limited by two main difficulties. First, the production of preparative quantities of natively folded and purified RNA molecules is a long and tedious process. To facilitate this step, our laboratory has developed an RNA-affinity purification method using an ARiBo tag. The second limiting step comes from the extensive signal overlap detected on NMR spectra of large RNA molecules. This overlap is proportional to the length of the RNA, which often prevents high-resolution structure determination of RNAs larger than 15 kDa. To solve this problem, specific isotopic labeling of RNAs can now be achieved. However, existing labeling protocols are expensive, require several weeks of laboratory manipulations and usually provide relatively low yields. This thesis provides an alternative strategy to achieve specific isotopic labeling of RNA molecules, based on the ARiBo tag affinity purification technique. The protocol includes the separation and the purification of isotopically labeled nucleotides, an enzymatic ligation step performed on a solid support and the affinity purification of the RNA of interest, without any sequence restriction. This new strategy is a fast and efficient way to label functional RNAs isotopically and should facilitate NMR structure determination of large RNAs.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
The 90 kDa Heat Shock Protein (Hsp90) is a chaperone protein responsible for regulating the activity of hundreds of structurally diverse client proteins in the cytosol, by providing assistance to their correct folding. In tumours Hsp90 is frequently found to be overexpressed assisting metastable proteins to remain active by stabilizing their conformation and helping them evade the biological degradation mechanisms. Over the past two decades, Hsp90 has become the focus of intense research activity as a possible target in new cancer therapies with drug candidates entering clinical trials targeting the well-known NTD of the protein, but these drugs have generally unfavourable pharmacokinetics and high potential for rebound adverse reactions. In the last few years, a new putative binding site at the CTD was found, explaining the antiproliferative activity of compounds such as the retired antibiotic novobiocin, but its structure hasn’t yet been clarified, hindering the efforts to develop new, safer and more effective, therapies. In this report, an attempt is made at the synthesis of a photoaffinity labelling molecular probe, to be used in a mass spectrometry assisted structural characterization of the CTD binding site, based on the current SAR models available for Hsp90 CTD inhibitors, derived from novobiocin.
A proteína de choque térmico de 90 kDa (Hsp90) é uma proteína chaperona, responsável pela regulação de grande variedade de proteínas citoplasmáticas, através da assistência ao seu correto folding. A expressão desta proteína está por vezes aumentada em certos tipos de tumores, onde, através da sua ação, permite que proteínas meta-estáveis se mantenham ativas e escapem aos mecanismos de degradação biológicos. Com efeito, a Hsp90 tornou-se desde há duas décadas um interessante foco de estudo como possível alvo para uma nova geração de fármacos antitumorais, existindo já fármacos em ensaios clínicos. Estes atuam sobre o já conhecido NTD, contudo apesar de representarem um avanço considerável em termos médicos, estas moléculas apresentam geralmente farmacocinética desfavorável e um efeito secundário preocupante de poderem provocar resposta paradoxal. Foi recentemente identificado um novo local de ação ao nível do CTD que poderá representar um alvo terapêutico promissor para uma nova geração de fármacos mais eficazes e seguros, mas o desenvolvimento de moléculas direcionadas a este alvo tem sido lento uma vez que ainda não foi possível esclarecer a sua estrutura. Neste relatório descreve-se a tentativa de sintetizar uma nova molécula para ser utilizada como sonda de marcação por foto-afinidade numa experiência, assistida por espectrometria de massa, de identificação estrutural da cadeia polipeptídica do local de ação do CTD. O design desta sonda tem por base a novobiocina, como inibidor deste local mais estudado, e os modelos de SAR já existentes.