The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in virulence of the whooping cough agent Bordetella pertussis. It translocates an AC enzyme into cytosol of CD11b+ phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming cation-selective pores. The goal of my diploma thesis was an analysis of the mechanism of interaction of the segment linking the invasive adenylate cyclase domain and the RTX hemolysin moiety of CyaA with target membrane. Our data show that the segment linking the AC to the hydrophobic domain of CyaA is directly involved in the interaction of the toxin with the membrane and controls the formation of small cationt-selective pores. Our results generate new knowledge that will be of relevance to the entire field of toxin biology and will enable the design of improved CyaA- based vaccines. Keywords: Bordetella pertussis, adenylate cyclase toxin, membrane translocation, pore- forming activity, black lipid bilayers, liposomes

Les bicouches lipidiques constituent l’architecture socle des membranes biologiques et l’environnement bidimensionnel de leurs protéines. Ancrées sur une interface hydrophile hydratée, ces systèmes conservent leur fluidité et sont localisés durablement près d’un substrat. Dans ce domaine, nous avons développé une stratégie de fonctionnalisation rapide, peu coûteuse et versatile, permettant la formation d’une membrane biomimétique fluide, ancrée sur des substrats conducteurs spécifiquement conçus pour son étude structurale et dynamique. La chimie de surface proposée forme une liaison covalente forte entre le substrat et des molécules commerciales amphiphiles (Brij, etc), utilisées comme système ancre-harpon. L’extrémité hydrophile (coté ancre) possédant un alcool primaire peu réactif est engagée sur une première couche organique par substitution nucléophile. L’autre extrémité hydrophobe (l’harpon) peut s’insérer dans la membrane et la stabiliser. Un mélange adapté, de ces molécules ancre-harpon avec d’autres purement hydrophiles (PEG, etc), apporte l’hydratation et la densité d’ancres nécessaire à l’interface pour maintenir la membrane éloignée du substrat, permettant ainsi l’intégration de protéines et le transport ionique à travers la membrane. Grâce au support conducteur, la dynamique des ions face aux membranes peut être étudiée par spectroscopie d’impédance électrochimique. Sa faible rugosité et semi-transparence permettent aussi l’utilisation de nombreuses autres techniques dont les microscopies optiques, exaltées ou de fluorescence. Localisées sur une électrode, ces bicouches ancrées s’ouvrent également aux applications biotechnologiques. / Lipid bilayers are the structural backbone of biological membranes and provide a two-dimensional environment for proteins. Tethered on a hydrophilic substrate, these biomimetic models are fluid, long-term stable and localized. In this regard, we propose a direct, cheap and versatile strategy of surface functionalization to tether membranes on a substrate adapted to their structural and dynamics study. The process is based on the functionalization of any flat metal thin film by the covalent binding of commercial surfactant molecules (Brij, …) as “anchor-harpoons”-like systems. Most of these molecules possess unresponsive –OH terminated groups on their hydrophilic moiety (anchor) that can bind a first organic layer by nucleophilic substitution. The opposite hydrophobic tail (harpoon) of the molecule can insert into the membrane and make it stable. An ideal mixing ratio of anchor-harpoons molecules with purely hydrophilic ones (PEG, …), provides the required hydration and density of anchors to the interface for tethering fluid membranes away from the substrate. A few nanometers distance enable ionic flows through the membrane and protein inclusion. The substrate conductivity enables studying ion dynamics facing the membrane by means of electrochemical impedance spectroscopy. Flatness and semi-transparency of the conductor opens the route to many other techniques’ including exalted light microscopy or fluorescence. Localized on electrodes, tethered bilayers further provide a biomimetic model and a support for biotechnology applications.

No description available.

Výměnná anizotropie je zajímavý fyzikální jev vznikající na rozhraní antiferomagnetických (AF) a feromagnetických (FM) materiálů, který již je široce používán v elektronickém průmyslu a magnetickém záznamu. Přestože byl tento jev dlouhou dobu intenzivně studován, jeho přesný mechanizmus zatím nebyl uspokojivě vysvětlen. V této práci je představen přehled studií dokumentujících výměnnou anizotropii v tenkých dvojvrstvách, včetně experimentálních výsledků a teoretických modelů. Experimentální úkoly této diplomové práce zahrnovaly jak výrobu, tak měření různých modelových systémů vykazujících výměnnou anizotropii. Dvojvrstva Fe/FeRh, kde vrstva FeRh prochází fázovou přeměnou z AF fáze na FM fázi při 360 K, poskytuje možnost nastavení parametrů výměnné anizotropie. Dále byly zkoumány účinky výměnné anizotropie a tvarové anizotropie v mikrostrukturách Fe/FeRh. Konečně, přítomnost výměnné anizotropie byla zkoumána mezi FM a AF fází koexistujícími během fázové přeměny v nanodrátech FeRh. Vzorky byly vyrobeny pomocí magnetronového naprašování a elektronové litografie. Všechny prezentované systémy byly analyzovány pomocí magnetooptické Kerrovy mikroskopie. Výměnná anizotropie byla úspěšně nalezena v systému Fe/FeRh, přičemž její velikost byla téměř identická co do rozsahu i orientace s výsledky v literatuře, přestože námi vyrobená dvojvrstva měla horší kvalitu FM-AF rozhraní. Bylo také prokázáno, že v tomto systému existuje tzv. tréninkový efekt (Training effect), což je výrazným důkazem existence výměnné anizotropie. U nanodrátů bylo změřena významná výměnná anizotropie mezi koexistujícími fázemi FM a AF během fázové přeměny.

This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.

Self-organization of regular surface pattern under ion beam erosion was described in detail by Navez in 1962. Several years later in 1986 Bradley and Harper (BH) published the first self-consistent theory on this phenomenon based on the competition of surface roughening described by Sigmund’s sputter theory and surface smoothing by Mullins-Herring diffusion. Many papers that followed BH theory introduced other processes responsible for the surface patterning e.g. viscous flow, redeposition, phase separation, preferential sputtering, etc. The present understanding is still not sufficient to specify the dominant driving forces responsible for self-organization. 3D atomistic simulations can improve the understanding by reproducing the pattern formation with the detailed microscopic description of the driving forces. 2D simulations published so far can contribute to this understanding only partially. A novel program package for 3D atomistic simulations called trider (TRansport of Ions in matter with DEfect Relaxation), which unifies full collision cascade simulation with atomistic relaxation processes, has been developed. The collision cascades are provided by simulations based on the Binary Collision Approximation, and the relaxation processes are simulated with the 3D lattice kinetic Monte-Carlo method. This allows, without any phenomenological model, a full 3D atomistic description on experimental spatiotemporal scales. Recently discussed new mechanisms of surface patterning like ballistic mass drift or the dependence of the local morphology on sputtering yield are inherently included in our atomistic approach. The atomistic 3D simulations do not depend so much on experimental assumptions like reported 2D simulations or continuum theories. The 3D computer experiments can even be considered as ’cleanest’ possible experiments for checking continuum theories. This work aims mainly at the methodology of a novel atomistic approach, showing that: (i) In general, sputtering is not the dominant driving force responsible for the ripple formation. Processes like bulk and surface defect kinetics dominate the surface morphology evolution. Only at grazing incidence the sputtering has been found to be a direct cause of the ripple formation. Bradley and Harper theory fails in explaining the ripple dynamics because it is based on the second-order-effect ‘sputtering’. However, taking into account the new mechanisms, a ‘Bradley-Harper equation’ with redefined parameters can be derived, which describes pattern formation satisfactorily. (ii) Kinetics of (bulk) defects has been revealed as the dominating driving force of pattern formation. Constantly created defects within the collision cascade, are responsible for local surface topography fluctuation and cause surface mass currents. The mass currents smooth the surface at normal and close to normal ion incidence angles, while ripples appear first at θ ≥ 40°. The evolution of bimetallic interfaces under ion irradiation is another application of trider described in this thesis. The collisional mixing is in competition with diffusion and phase separation. The irradiation with He+ ions is studied for two extreme cases of bimetals: (i) Irradiation of interfaces formed by immiscible elements, here Al and Pb. Ballistic interface mixing is accompanied by phase separation. Al and Pb nanoclusters show a self-ordering (banding) parallel to the interface. (ii) Irradiation of interfaces by intermetallics forming species, here Pt and Co. Well-ordered layers of phases of intermetallics appear in the sequence Pt/Pt3Co/PtCo/PtCo3/Co. The trider program package has been proven to be an appropriate technique providing a complete picture of mixing mechanisms.

Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte. Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen. Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden. Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center. In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation. Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved. In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.

No description available.

Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K⁺ channels. Molecular models were built of the inwardly rectifying K⁺ channel Kir2.2, the bacterial homolog K⁺ channel KirBac3.1, and the twin pore (K2P) K⁺ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K⁺ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K⁺ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K⁺ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.

Les parasporines sont des toxines Cry du bacille de Thuringe actives contre des cellules tumorales. Ce travail montre que la parasporine PS1Aa2 (Cry31Aa2) forme des pores dans des membranes artificielles, comme de nombreuses toxines Cry. Ceux-ci ont plusieurs niveaux de conductance dont les plus fréquents étaient de 11, 16 et 21 pS dans une solution de 150 mM KCl. Nos résultats de microspectrofluorométrie avec la sonde Fura-2 montrent que la présence de la PS1Aa2 peut produire des augmentations du calcium intracellulaire, la plupart du temps sous la forme d’oscillations calciques et parfois des augmentations soutenues. Ces réponses ont été observées en présence et en absence de calcium extracellulaire, dans les lignées tumorales HeLa et HepG2 et dans la lignée non tumorale HEK 293. Bien que quelques études aient montré que le calcium semble intervenir dans leur mode d’action, de telles oscillations calciques n’ont jamais été décrites auparavant pour des toxines Cry. Les expériences ont dû être faites à des concentrations beaucoup plus élevées de toxine que prévues sur la base des résultats publiés de cytotoxicité. Malgré la présence des fragments identifiés auparavant comme actifs, sa faible efficacité semble liée à la présence d’ADN dans les préparations qui entraîne la précipitation de la protéine. Les travaux futurs sur cette toxine seraient donc grandement facilités par une amélioration de sa méthode de préparation. / Parasporins are Cry toxins from Bacillus thuringiensis that are active against tumor cells. This work shows that parasporin PS1Aa2 (Cry31Aa2) forms pores in artificial membranes like many Cry toxins. These pores have several levels of conductance; the most frequently seen in 150 mM KCl solutions were of 11, 16 and 21 pS. Our microspectrofluorometric results with the Fura-2 probe showed that the presence of PS1Aa2 can induce changes in intracellular calcium levels, most often in the form of calcium oscillations and sometimes as sustained increases. Such responses were observed in the presence and absence of extracellular calcium, with the tumor cell lines HeLa and HepG2, and with the non-tumorous cell line HEK 293. Calcium oscillations have not been described previously for Cry toxins even though some studies have shown that calcium appears to be involved in their mode of action. Our experiments required the use of much higher concentrations of toxin than suggested from the published cytotoxicity results. Despite the presence of fragments previously identified as active, its low efficacy appears to be related to the presence of DNA in the preparations causing the protein to precipitate. Future work on this toxin would therefore be greatly facilitated by an improvement in its method of preparation.