The thesis entitled “Folding studies on Peanut Agglutinin: A lectin with an unusual quaternary structure” deals with the several aspects of the folding of the tetrameric legume lectin Peanut Agglutinin (PNA). PNA is a well studied legume lectin and several interesting observations regarding its unfolding have been published from our laboratory. The present thesis is an extension of the same work to enrich our knowledge about the folding behaviour of PNA. The thesis describes both experimental as well as theoretical insight on unfolding of PNA. Chapter 1 is a general discussion on lectins. Lectins are carbohydrate binding proteins of non immune source. Lectins are generally found in all type of organisms- plants, animals as well as micro-organisms. Among the plant lectins “legume lectin” is a very well studied system. Legume lectins share a general tertiary structural fold; “jelly roll fold” while they vary in their quaternary structure. Thus they can be considered as “natural mutants” in the context of quaternary structure. The origin of the lectins, structure and sugar specificity have been discussed with emphasis on legume lectin family. Chapter 2 describes the thermodynamics related to the urea induced denaturation of PNA. PNA shows a very interesting unfolding profile, populating one molten globule like intermediate during thermal as well as chaotrope induced denaturation. The molten globule like intermediate loses most of its tertiary structure but retains sufficient secondary structure. Surprisingly, the molten globule like state retains its carbohydrate binding specificity like the native PNA. A model has been developed to fit the chaotrope induced three state denaturation profile of PNA. The model considers the tetramer to dissociate to monomeric intermediate, which in turn dissociates to complete denatured state. All the relevant thermodynamic parameters (∆G, ∆Cp, Tg) associated in the denaturation process have been extracted. The tetramer is found to be ~30 kcal/Mole more stable compared to the intermediate and the intermediate is ~8 kcal/Mole more stable compared to the denatured. The denaturation process has been followed by the changes in hydrodynamic radii by dynamic light scattering (DLS). The profile of change in hydrodynamic radius and the % intensity clearly identify the generation of two species simultaneously. The analysis shows that the intermediate is ~40 % unfolded in nature. Thus this chapter deals with the detailed study of thermodynamics and dynamic light scattering study of the urea induced denaturation of PNA. Chapter 3 deals with the effect of 2, 2, 2 - trifluoroethanol (TFE) on the structure of PNA at two different pH. TFE is a well known co-solvent and is widely used to induce α- helical structure in a protein. The secondary structures induced by TFE are assumed to reflect conformations that prevail during early stages of protein folding. Thus it was quite interesting to notice the structural changes induced by TFE. The effect of TFE has been studied at two different pH- neutral pH of 7.4 and acidic pH 2.5. The  structure of the protein is accentuated in the presence of TFE at low concentration at both the pH. TFE induces α-helical structure from 40 % (v/v) concentration onwards at both the pH. TFE at 15 % concentration induces a molten globule like structure at low pH. The quenching of acrylamide suggests that the protein at low pH and 15 % TFE concentration has a more compact structure compared to the protein at low pH in absence of TFE as well as 6M guanidine hydrochloride (GdnHCl). Further studies of hydrodynamic radii by dynamic light scattering (DLS) also reveal that the protein undergoes some kind of compaction in presence of 15 % TFE at low pH. The induction of this type of molten globule like state at neutral pH has not been observed. Chapter 4 describes the molecular dynamics simulation of deoligomerization of PNA. The native PNA (PDB code 2PEL), excluding any ligand and metal ions has been simulated at 300 K, 400 K, 500 K and 600 K for 500 ps. The overall destabilisation has been followed by root mean square deviation (RMSD), the radius of gyration (Rg) and the solvent accessible surface area (ASA), while the atomistic details are revealed by residue wise RMSD (RRMS), hydrogen bonds and cluster analysis. The protein shows a quite a dramatic change in RMSD and radius of gyration profile at 600 K. RRMS shows that the residues belonging to the loops, mainly in the metal binding site show quite high flexibility. The relative change in average accessible surface area reveals that the primary core of the protein is exposed at 600 K while it is well buried till 500 K. The hydrogen bond analysis clearly shows that with increase in temperature number of hydrogen bonds starts decreasing. Mainly the hydrogen bonds involving side chain interactions are broken. Surprisingly, not all the monomers behave similarly. Monomers C and D are more perturbed compared to monomers A and B. The asymmetry in the interfaces of the monomers may be the key reason for it. The change in the interfaces has been probed by hydrogen bond analysis and cluster analysis. The GSIV type interfaces (A-D and B-C) have been found out to be the most dynamic in nature compared to the other two interfaces. Thus, this chapter reveals the early stage of unfolding of PNA, where perturbation in secondary and tertiary structural level is quite prominent but the interfaces are still holding weakly and are not completely dissociated. Chapter 5 is the continuation of the molecular dynamics simulation on unfolding of PNA, where the effect of metal ions has been illustrated. The monomeric PNA has been simulated to compare its dynamics with the tetramer. The metal binding loop (125-135) becomes unstable and opens up for the monomer even at 300 K after 800 ps. The monomer at 600 K is completely disorganized. The instability of the metal binding loop of the monomer triggers the urge to study the simulation in presence of metal ions (Ca2+ and Mn2+). The monomer bound with metal ions shows steady fluctuation at 300 K. Binding of metal ions seems to bring stability even at 600 K. Surprisingly binding of metal ions to the metal binding site not only stabilises the metal binding loop but also stabilises residues at back beta sheet which are involved in oligomerization. Hence, another simulation of the tetramer at 600 K bound with metal ions has been done. It has been shown that binding of metal ions increases the stability of the protein without altering the denaturation pathway. Appendix A describes a completely different study from PNA. The initial spectral and kinetic characterization of 7, 8- Diaminopelargonic acid Synthase (DAPA Synthase) has been done from Mycobacterium tuberculosis. The DAPA Synthase gene has been cloned earlier in our laboratory and the same has been used for further studies. This is a well known pyridoxal-5′ phosphate (PLP) dependent enzyme, which converts 8- Amino-7-oxopelargonic Acid (KAPA) to 7, 8-Diaminopelargonic Acid (DAPA) in the second step of biotin biosynthesis. DAPA Synthase uses S-adenosylmethionine (SAM) and KAPA as substrate. The first half of the enzymatic reaction has been followed spectroscopically, both by steady state and stopped flow. The enzyme seems to undergo change in conformation as evident from fluorescence and circular dichroism study. The Km value has been determined using bioassay technique. The detailed characterization of the enzyme has been described in this chapter.

No description available.

Le présent projet vise à analyser l'influence de la multivalence dans les interactions sucres-lectines. En collaboration avec des équipes externes, une étude par microscopie à force atomique (AFM) de l'interaction entre des ligands synthétiques de différentes valences et leurs lectines spécifiques a été entreprise. Dans le cadre de cette étude, une première caractérisation fondamentale de l'interaction sucre-lectine a été menée. Cette caractérisation concerne plus particulièrement l'influence de la multivalence sur les forces d'adhésion et la dynamique de l'interaction entre les ligands synthétiques multivalents et une lectine modèle, la lectine d'arachide PNA. Une seconde caractérisation, d'aspect plus appliqué, concerne l'utilisation des ligands synthétiques multivalents dans une approche thérapeutique antiadhésive pour le traitement des infections urinaires chroniques dues à Escherichia coli uropathogène (UPEC). Le caractère innovant des ligands (obtenus par une synthèse chimique rationnelle) ainsi que l'approche utilisée pour caractériser leurs interactions avec les lectines à l'échelle moléculaire par AFM témoigne de l'originalité du projet.

Lipidmembranen ermöglichen die räumliche Anordnung von Biomolekülen. Einerseits repräsentieren Lipidvesikel Kompartimente zur Aufrechterhaltung chemischer Milieus und dienen der Verkapselung verschiedenster Substanzen. Anderseits stellen inhomogene Membranen Matrizen für eine laterale Organisation von Membrankomponenten dar. In der vorliegenden Arbeit wurden lipophile Nukleinsäuren zum Aufbau kompartimentalisierter Strukturen auf der Basis von Lipidmembranen benutzt, erstens, für die geordnete, dreidimensionale Assemblierung von Vesikeln, zweitens, für eine spezifische Funktionalisierung inhomogener Lipidmembranen. Definierte Schichten stabiler Lipidvesikel wurden auf „layer-by-layer“ beschichteten Silikapartikeln angeordnet. Mit Hilfe einer optischen Pinzette wurde der gerichtete Transport der mit Vesikeln beschichteten Partikel demonstriert. Moleküle konnten in den Vesikeln verkapselt und bei Bedarf vor Ort freigesetzt werden. Zudem wurde die kontrollierte Fusion der immobilisierten Veskel gezeigt, die eine Durchmischung von verschiedenen Membrankomponenten zur Folge hatte. Lipophile Nukleinsäuren wurden in die Membranen von lipiddomänenbildenden Vesikeln inkorporiert. Cholesterolbasierte DNS verteilte sich hierbei homogen über die gesamte Membran. Palmitoylierte Peptid-Nukleinsäure konzentrierte sich hingegen in der flüssig-geordneten Phase von flüssig-flüssig phasenseparierten Membranen, welche sogenannten Lipid Rafts in Zellmembranen ähnelt. Mittels der palmitoylierten Peptid-Nukleinsäure und tocopherolmodifizierter DNS wurden lateral inhomogene Membranen domänenspezifisch funktionalisiert. Beide Konstrukte konnten temperaturabhängig vermischt und separiert werden. / Lipid membranes are versatile tools for the spatial organization of biomolecules. On one hand, lipid vesicles represent enclosed compartments to maintain chemical environments and allow the efficient entrapment of substances. On the other hand, lateral inhomogeneous membranes provide the two dimensional sorting of membrane-bound compounds. In this work, lipophilic nucleic acids were used to build multicompartment systems based on lipid membranes by the controlled assembly of vesicles and the domain specific functionalization of inhomogeneous membranes. Three dimensional architectures of vesicles were formed by the sequential assembly of vesicles on layer-by-layer coated particles. Upon binding of the vesicles to the particles the vesicles remained stable – they did not fuse neither became leaky. Molecules could be entrapped inside the vesicles and released on demand. It was shown that the vesicles assembled on a particle can be transported to a defined destiny using an optical tweezer. Thus, the targeted delivery and the release of encapsulated molecules on site was achieved. It was also shown that vesicles immobilized on the particles can be fused by remote control, resulting in a mixing of membrane associated compounds. Different lipophilic nucleic acids were arranged in two dimensional patterns by incorporation into domain-forming vesicles. Cholesterol-modified DNA revealed an equal distribution to both domains in liquid-liquid phase-separated membranes, whereas palmitoylated peptide nucleic acid partitioned into the liquid-ordered domain, which resembles lipid rafts of cellular membranes. Using the palmitoylated peptide nucleic acid and tocopherol-modified DNA both domains of liquid-liquid phase-separated vesicles were functionalized with different DNA recognitions sites. Both constructs could be mixed and separated by temperature control.

L'infection par le virus de l'hépatite C (VHC) demeure une cause majeure de cirrhose et de carcinome hépatocellulaire. Compte tenu d'une efficacité encore insuffisante des thérapies actuelles (55% de succès en moyenne), le développement de nouvelles stratégies antivirales reste une priorité. Dans ce contexte, nos travaux ont porté sur l'évaluation de nouveaux variants de l'interféron alpha dans un modèle de réplicon VHC. Nous avons pu identifier un variant, GEA007.1 qui présente une activité anti-VHC supérieure à celle de l'IFN alpha-2b utilisé en clinique associé à une plus grande efficacité de transduction du signal. Nous avons également évalué une approche antisens avec des molécules de type Peptide Nucleic Acids (PNA) et montré qu'elle permettait d'inhiber la traduction in vitro du VHC par blocage de la région interne d'entrée du ribosome (IRES), de manière plus efficace et spécifique qu'une approche antisens avec des molécules de premières générations. Une telle stratégie se justifie par la conservation de la cible qui pourrait se révéler utile avec l'utilisation prochaine des anti-protéases et anti-polymérases du VHC et le risque inhérent d'apparition de résistances au traitement. L'ensemble de ces résultats montre que l'amélioration de la bithérapie actuelle est possible et que de nouvelles approches thérapeutiques fonctionnent.

Ce travail s’inscrit dans le cadre de la recherche relative au développement de biosenseurs à ADN électrochimiques. Des aspects fondamentaux, ainsi que des aspects d’application de la détection d’hybridation d’ADN sont envisagés.<p>Dans un premier temps, le comportement interfacial et le processus d’hybridation d’oligonucléotides d’ADN linéaires et ADN hairpin (structure en épingle à cheveux) nonmarqués sont étudiés en formant des monocouches auto-assemblées mixtes de monobrins d’ADN (ssADN) thiolés et d’un hydroxyalcanethiol (4-mercaptobutan-1-ol) par coadsorption spontanée sur des électrodes d’or polycristallin. L’immobilisation de monocouches mixtes ssADN/MCB est caractérisée par voie électrochimique et par spectroscopie des photoélectrons X. Des mesures de chronocoulométrie, en présence de [Ru(NH3)6]3+ (RuHex), permettent de déterminer la quantité d’ADN dans la monocouche mixte formée. Les résultats montrent que l’excès superficiel d’ADN linéaire est plus important que l’excès superficiel d’ADN hairpin sous des conditions de formation identiques.<p>La réaction de reconnaissance moléculaire d’hybridation est détectée par des mesures d’impédance en présence de [Fe(CN)6]3-/4-. L’hybridation se traduit dans le cas de l’ADN linéaire par une augmentation de la résistance au transfert d’électron Rct tandis que dans le cas de l’ADN hairpin, Rct diminue. Ces différences sont dues au plus faible recouvrement et au changement de conformation des molécules d’ADN hairpin lors de l’hybridation. Des mesures de réflectivité de neutrons nous ont permis de mettre en évidence l’augmentation de l’épaisseur du film d’ADN hairpin et de confirmer le changement conformationel ces sondes lors de la reconnaissance moléculaire.<p>Dans la seconde partie, nous présentons une nouvelle méthode électrochimique de détection d’hybridation, basée sur les interactions électrostatiques entre le complexe cationique RuHex et les groupements phosphates de l’ADN. Afin d’améliorer la détection des molécules de PNA (peptide nucleic acid) ont été immobilisées comme sondes de reconnaissance moléculaire. Après hybridation des sondes PNA avec le brin complémentaire, RuHex s’adsorbe sur l’ADN hybridé et un signal de réduction de ces complexes redox, enregistré par voltampérométrie alternative, constitue une signature claire de l’hybridation d’ADN à l’interface modifiée. Les interactions RuHex/PNA-ADN ont été étudiées. La constante d’adsorption de RuHex sur l’électrode modifiée PNA/MCB après hybridation est évaluée à 2,9 (±0,3) 105 M-1 en milieu Tris-HCl 0,01M, selon une isotherme de Langmuir.<p>Les performances analytiques de la méthode de détection (sensibilité, sélectivité et reproductibilité) ont été évaluées et optimisées pour la détection des séquences d’ADN du gène de l’ARNr 23S d’Helicobacter pylori. La méthode de détection électrochimique présentée est assez sélective pour permettre de discriminer les mutations ponctuelles A2143G et A2144C de la séquence de type sauvage. La diminution significative des signaux d’admittance enregistrés en présence des séquences mutées est attribuée à la capacité accrue de discrimination de mutations ponctuelles des molécules PNA.<p>La réponse de détection est linéaire en fonction du logarithme de la concentration de la cible d’ADN sur plus de quatre ordres de grandeur (10-6 M à 10-10 M). La limite de détection de l’oligonucléotide d’ADN complémentaire de 80 pM est très bonne. La méthode a été appliquée avec succès à la détection de fragments PCR complémentaires de 100 et 400 paires de bases, amplifiés à partir de souches SS1 d’H.pylori. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / The frequency and magnitude of floods in the midcontinental United States have increased in recent decades due to changing precipitation patterns as global temperatures rise. These trends pose major social and economic risks to the region, which is home to tens of millions of Americans and a global agricultural center. It is therefore critical to understand if current fluvial dynamics are within the scope of past fluvial-climate relationships, or if they represent a novel response to recent climate and land-use changes. Presented is a 1600-year-long flood frequency record for the moderately sized (~29,400 km2 watershed) White River, Indiana. Flood frequencies were determined using 14C-based sediment accumulation rates at Half Moon Pond, an oxbow lake on the lower White River’s floodplain. Comparison with regional paleoclimate data shows that White River flooding was frequent when atmospheric circulation resembled the negative mode of the Pacific-North American (PNA) teleconnection, particularly during the Medieval Climate Anomaly (950-1250 CE) and the Current Warm Period (last ~150 years). During these times, the regional climate was dominated by warm-season precipitation originating from the Gulf of Mexico. Conversely, White River flooding was less frequent during the Little Ice Age (1250-1800 CE) when cold-season precipitation from the North Pacific/Arctic dominated (+PNA-like conditions). The pre-1790 CE White River flood history was antiphased with reconstructed Ohio River flood frequencies from southern Illinois. This dynamic is consistent with discharge in small to moderate sized watersheds being sensitive to rainstorm runoff and large watersheds being sensitive to snowmelt runoff. After 1790 CE, flooding frequencies of both river systems increased to their highest levels, despite a shift to -PNA-like conditions. This change was likely due to extensive Euro-American land-clearance, which increased runoff/erosion by reducing evapotranspiration, interception, and infiltration. While the White River responded strongly to climatic conditions in the past that were similar to present conditions (-PNA-like conditions), recent land-use practices have amplified the effects of the current hydroclimate. Since a warming climate is expected to increase regional average precipitation and extreme rainfall events, and that landscape modifications have lowered surface resilience to hydroclimate events, flooding will likely become more frequent in the coming decades.

No description available.

Diese Arbeit ist Teil eines Weges, der die Polarisationsdynamik von Polynukleinsäure-Helices (DNA) zum Ziel hat. Die Dynamik kann im Prinzip von innen beobachtet werden, über Femtosekunden-zeitaufgelöste optische Spektroskopie eines Farbstoff-Moleküls anstelle einer Nukleinbase. Der optische Chromophor soll als solvatochrome Sonde der DNA Umgebung funktionieren. Dafür wurde 2-Amino-7-Nitro-Fluoren (ANF) gewählt, welches ideale solvatochrome Eigenschaften hat. Bevor der Einbau des Sondenmoleküls erfolgt, müssen dessen photophysikalischen Eigenschaften in reinen Lösungsmitteln und in Lösungsmittel-Mischungen weitgehend verstanden sein. Dieses Thema bestimmt den größeren Teil der Arbeit. ANF, aliphatische Derivate, und das entsprechende Nukleosid wurden synthesitsiert. Ihre thermodynamischen Eigenschaften, optischen Spektren, und ps Fluoreszenzlebensdauern werden für eine Reihe von Lösungsmitteln beschrieben. Präferentielle Solvatation in wässrigen Mischungen wird mit optischer Absorptions- und 1H NMR-Spektroskopie untersucht. Inclusionskomplexe mit Cyclodextrinen werden ebenfalls charakterisiert. Auf diese "klassischen" Studien folgt die fs-transiente optische Absorptionsspektroskopie von ANF und Derivaten. Die transienten Spektren werden zeilegt und die Banden für stimulierte Emission als Funktion der Zeit in den meisten Fällen erhalten. Damit erhält man eine Solvatations-Relaxationsfunktionen C(t) für die meisten Lösungsmittel (50 fs Zeitauflösung). Unterschiede zu bekannten Relaxationsfunktionen für die vieluntersuchte Sonde Coumarin 153 werden diskutiert. Wichtig ist, daß derart C(t) für ein ANF Nukleosid in Wasser existiert. Dies kann mit einer simulierten Kurve für ANF in DNA-Helices verglichen werden. Es folgt, dass gestapelte Chromophore im Innern dynamisch von jenen unterschieden werden können, die in die wässrige Umgebung herausgedreht sind. Der Cyaninfarbstoff Thiazol Orange (TO) wird von anderen Gruppen verwendet, um die Hybridisierung von DNA-Strängen zu verfolgen. Die Fluoreszenz von TO wird durch schnelle Isomerisation im S1 Zustand stark gelöscht. In dieser Arbeit wird die entsprechende innermolekulare Verdrillung mit fs-transienter Absorptionsspektroskopie und mit fs-zeitaufgelöster Fluoreszenzspektroskopie (Summenfrequenz-Erzeugung) zum ersten Mal zusammenhängend untersucht. Transiente Spektren in Lösung werden mit stationären Spektren in verschiedenen PNA/DNA Duplex-Konstrukten verglichen. Eine hochfrequente Schwingungsmode ist bei der strukturellen Reorganisation von TO nach S0 to S1 Anregung beteiligt. Zunehmende Aktivität begleitet die Verdrillung. Deshalb sollte die Form der stationären Fluoreszenzbande dazu geeignet sein, das verfügbare freie Volumen in DNA-Konstrukten abzuschätzen. / This work is part of an effort to observe the polarization dynamics of duplex polynucleic acids from the inside, through femtosecond time-resolved optical spectroscopy of a dye molecule which has been stacked into the structure at a well-defined site. The dye chromophore should act as a solvatochromic probe of its DNA environment. For a probe, 2-amino-7-nitro fluorene (ANF) had been selected since it has ideal solvatochromic properties. Before being linked into DNA, its photophysical properties in liquids and liquid mixtures should be understood. The larger part of this thesis deals with this topic. ANF, aliphatic derivatives, and the nucleoside were synthesized. Their thermodynamic properties, optical spectra, and ps fluorescence decay are examined in a wide range of solvents. For preferential solvation in aqueous mixtures, absorption results are compared with 1H NMR spectra. Inclusion properties were also explored. Femtosecond transient absorption spectroscopy of ANF and derivatives follows after these "classical" studies. The transient spectra are analyzed and, in most cases, the stimulated-emission band can be obtained as function of time. Ultrafast solvation relaxation functions (50 fs resolution) for many solvents are constructed from these data and discussed by comparison with a well-studied probe, Coumarin 153. Most importantly, the solvation relaxation of the ANF nucleoside in water is obtained and compared with a simulated relaxation of the chromophore in a DNA duplex. It follows that chromophores stacked inside DNA can be distinguished from chromophores which extend into the aqueous boundary layer. The cyanine dye Thiazole Orange (TO) is used by other groups to monitor the hybridisation of DNA strands. Its fluorescence is strongly quenched by intramolecular twisting in the excited state S1. In this thesis the twisting process is characterized by femtosecond transient absorption and fluorescence upconversion spectroscopy, combined, for the first time. The transient spectra are compared to stationary spectra in a PNA/DNA duplex where the degree of twisting is restricted. A high-frequency mode is shown to play an important role in the structural reorganisation of TO following S0 to S1 excitation. Its vibrational activity is correlated with twisting. Therefore the shape of the emission band may be used to estimate the available volume in DNA constructs.

No description available.