The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

The Arabidopsis thaliana glycosyltransferase UGT72B1 is one of many enzymes which catalyze the reaction oflinking a glucose moiety from UDP-glucose to an acceptor molecule, in this case a chloroaniline or a chlorophenol. This is part of a detoxification system of the plant cell, similar to that in humans where a glucuronosyltransferases are enabling drug metabolism. It would be of interest to investigate the activity of the human enzyme towards different pharmaceuticals and determine the effect the linkage of glucose has to properties of the compounds. However, the human enzymes are membrane proteins and thus difficult to purify and crystallize. Here, an attempt was made to instead change the substrate specificity of UGT72B1 from UDPglucose to UDP-glucuronic acid. Combination of the four point mutations G18S, P139R, W367S and AG387ED were introduced in UGT72B1. However, no UDP-glucuronic acid activity was obtained. Single mutants W367S and AG387ED retained similar activity as of the wildtype while P139R had highly reduced activity and G18S was not expressed at all. All other combinations of mutations resulted in even less activity. Four chimeric proteins were also constructed. They were combinations of the UGT72B1 and the human enzyme UGT2B4. These were all soluble proteins but no activity could be determined. / Glykosyltransferaset UGT72B1 från Arabidopsis thaliana är ett av många enzymer som katalyserar reaktionen där en glukosenhet från UDP-glukos länkas till en acceptormolekyl, i det här fallet en kloranilin eller en klorfenol. Det är en del av ett detoxifieringssytem i växtcellen, som liknar det i människan, där ett glukuronosyltransferas möjliggör nedbrytning av bl.a. läkemedel. Det vore intressant att kunna undersöka de humana enzymernas aktivitet mot olika läkemedel och även fastställa effekten glukoslänkningen har på dessa substansers egenskaper. De humana enzymerna är dock membranprotein och är därför svåra att rena fram och att kristallisera. Här har istället ett försök gjorts för att ändra substratspecificiteten hos UGT72B1 från UDP-glukos till UDP-glukuronsyra. Kombinationer av de fyra punktmutationerna G18S, P139R, W367S och AG387ED introducerades i UGT72B1. Ingen aktivitet med UDP-glukuronsyra erhölls dock. Enkelmutanterna W367S och AG387ED bibehöll liknande aktivitet som vildtypen, medan P139R hade starkt reducerad aktivitet och G18S uttrycktes inte alls. Alla andra kombinationer av mutationer resulterade i ännu lägre aktivitet. Fyra chimeriska proteiner konstruerades också. De skapades genom kombination av UGT72B1 och det humana enzymet UGT2B4. Dessa var alla lösliga proteiner men ingen av dem uppvisade någon aktivitet.

No description available.

The main focus of this project was to optimize a protocol for small molecule ligand co-purification from an in-vivo tissue source. For this purpose, I employed a transgenic zebrafish line called the pLT-gypsy, which expresses a fusion protein containing a tagged-NR LBD (Tiefenbach et al., 2010). The particular line I used to optimize the ligand identification protocol is the pLT-PPARγ zebrafish line, which expresses the tagged-PPARγ receptor's LBD (also called PPARγ-fusion protein). By using rosiglitazone (a known PPARγ ligand) as a positive control, I managed to optimize a protocol to purify the PPARγ-fusion protein and identify the co-purified ligand by mass spectrometry. This protocol can be used to identify the physiological/endogenous ligand for the PPARγ receptor as well as other orphan NRs. Compared to previous methods of ligand identification, this method allows for the identification of the ligand from the tissues where it is functional.

The main focus of this project was to optimize a protocol for small molecule ligand co-purification from an in-vivo tissue source. For this purpose, I employed a transgenic zebrafish line called the pLT-gypsy, which expresses a fusion protein containing a tagged-NR LBD (Tiefenbach et al., 2010). The particular line I used to optimize the ligand identification protocol is the pLT-PPARγ zebrafish line, which expresses the tagged-PPARγ receptor's LBD (also called PPARγ-fusion protein). By using rosiglitazone (a known PPARγ ligand) as a positive control, I managed to optimize a protocol to purify the PPARγ-fusion protein and identify the co-purified ligand by mass spectrometry. This protocol can be used to identify the physiological/endogenous ligand for the PPARγ receptor as well as other orphan NRs. Compared to previous methods of ligand identification, this method allows for the identification of the ligand from the tissues where it is functional.

L’hexokinase (HK) est la première enzyme du métabolisme des hexoses et catalyse la réaction qui permet aux hexoses d’entrer dans le pool des hexoses phosphates et donc par le fait même la glycolyse. Bien que le glucose soit son principal substrat, cette enzyme peut aussi phosphoryler le mannose et le fructose. Malgré son importance dans le métabolisme primaire, l’HK n’a jamais été purifiée à homogénéité sous forme native. Le but de ce travail était donc de purifier une isoforme d’HK à partir de tubercule de Solanum tuberosum et par la suite de caractériser ses propriétés cinétiques. Bien avant que je commence mon travail, un groupe de recherche avait déjà séparé et partiellement purifié trois isoformes d’HK de S. tuberosum. Un protocole d’extraction était donc disponible, mais l’HK ainsi extraite était peu stable d’où le besoin d’y apporter certaines modifications. En y ajoutant certains inhibiteurs de protéases ainsi qu’en modifiant les concentrations de certains éléments, le tampon d’extraction ainsi modifié a permis d’obtenir un extrait dont l’activité HK était stable pendant au moins 72h après l’extraction, en empêchant la dégradation. À l’aide du tampon d’extraction optimisé et d’une chromatographie sur colonne de butyl sépharose, il a été possible de séparer 4 isoformes d’HKs. Par la suite, une isoforme d’HK (HK1) a été purifiée à l’homogénéité à l’aide de 5 étapes de chromatographie supplémentaires. En plus de caractériser les propriétés cinétiques de cette enzyme, l’analyse de séquençage par MS/MS a permis de l’associer au produit du gène StHK1 de Solanum tuberosum. Avec une activité spécifique de 10.2 U/mg de protéine, il s’agit de l’HK purifiée avec l’activité spécifique la plus élevée jamais rapportée d’un tissu végétal.L’ensemble des informations recueillies lors de la purification de HK1 a ensuite été utilisée pour commencer la purification d’une deuxième isoforme (HK3). Ce travail a permis de donner des lignes directrices pour la purification de cette isoforme et certains résultats préliminaires sur sa caractérisation enzymatique. / Hexokinase (HK) catalyzes the first step of hexose metabolism by phosphorylating hexose to generate the corresponding hexose phosphate thereby allowing hexose entrance in glycolysis. Even though glucose is the main substrate, HK can also phosphorylate a broad spectrum of hexoses. Despite its importance this enzyme has never been purified to homogeneity in a native form. The aim of this work was therefore to purify this enzyme from Solanum tuberosum tubers and subsequently characterize its kinetic properties. Before I started this work, another group had already separated and partially purified 3 HK isoform from S. tuberosum. An extraction protocol was available but improvement was necessary since the extracted HK had little stability. By adding some protease inhibitors and by modifying the concentration of certain components in the extraction buffer we were able to obtain an extract with a HK activity stable for at least 72 h after extraction by preventing degradation. With this buffer and chromatography on butyl sepharose it was possible to separate 4 HK isoforms from S. tuberosum. After 5 more chromatographic steps, one HK isoform was purified to homogeneity (HK1). This enzyme was characterized and sequenced by MS/MS. We were able to associate this protein sequence with the gene product of StHK1 from S. tuberosum. With a specific activity of 10.2 U/mg of protein, this is the HK with the highest specific activity ever reported from a plant tissue. All the information gathered while purifying HK1 was used to undertake the purification of a second isoform (HK3). We were able to obtain preliminary results on its kinetic properties.

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles. / Abstract RNase P is a ribonucleo-protein complex (an RNA enzyme or ribozyme) that cleaves 5’ leader sequences of precursor tRNAs and a few other small RNAs. It occurs in all three domains of life, Bacteria, Archaea and Eukarya, with the latter containing distinct nuclear and organellar (mitochondrial or plastid) activities. In most instances, the complex contains a single, well-conserved RNA subunit that carries the active center of the enzyme. Yet, compare to bacterial and nuclear P RNA, most mtP RNAs are structurally highly reduced. The number of P proteins is highly variable: one in Bacteria, about four in Archaea, and ten in the cytoplasmic form of Eukarya. Much less is known in the case of mitochondria. MtRNase P is usually purified by using numerous separation steps that include unphysiological conditions, leading to complexes having a minimum number of subunits (e.g., in yeast and Aspergillus nidulans), that often loose their activity. Here, using BN PAGE, we have developed an enrichment procedure for A. nidulans mtRNase P that avoids some of the most disruptive conditions. The protein composition of active fractions was identified with LC/MS/MS, indicating that the RNase P holoenzyme is much larger than previously thought. Finally, the question of mtRNase P localization within mitochondria was investigated, by tracing its RNA subunit by RT PCR. We found that mtRNase P of A. nidulans is a predominantly membrane-attached enzyme; it is in part solubilized by detergents such as digitonin and Triton.