Colicin N and its thermolytic fragment induce phospholipid vesicle fusion

AbstractColicin N, a bacteriocin encoded on a plasmid belonging to the pore-forming class of colicins, induces phospholipid vesicle fusion at acidic pH as demonstrated by fluorescence resonance energy transfer. Its C-terminal thermolytic fragment has properties very similar to the native molecule. The fusion is protein concentration-dependent and is regulated by (a) group(s) with a pK of approximately 4.6. The physiological relevance of this characteristic common to all colicins tested so far is discussed

Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E. coli outer membrane

AbstractHomogeneous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NACl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the build-up of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking (SPT). Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3 mu m^2/s on the L-DPPC and 4.6 mu m^/s on the D-DPPC monolayers. In regions enriched with hydrolysis products the diffusion dropped to approx. 0.2 mu m^2/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between approx. 30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e. enzymes were simply reflected from the gel domains back into solution.Comment: 10 pages, 10 figure

A β Strand Lock Exchange for Signal Transduction in TonB-Dependent Transducers on the Basis of a Common Structural Motif

SummaryTransport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a β strand which interacts through a mixed four-stranded β sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded β sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a β strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe

pH-dependent Stability and Membrane Interaction of the Pore-forming Domain of Colicin A*

Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A

Interactions of small polypeptides with dimyristoylphosphatidylcholine monolayers: effect of size and hydrophobicity

The effects of size and hydrophobicity of small (molecular weights below 2,000) polypeptides on their predominantly hydrophobic interactions with a neutral phospholipid monolayer were studied. The changes in surface pressure were determined when various concentrations of Gly, Gly-Gly-Gly, -Ala, -Ala--Ala--Ala, -Ala-Gly-Gly-Gly-Gly, -Phe--Leu--Glu--Glu--Leu, adrenocorticotropic hormone fragments 1-10 (ACTH-(1-10)), porcine [beta]-lipotropin, [alpha]-endorphin and human fibrinopeptide A were injected under dimyristoylphosphatidylcholine (DMPC) monolayers at an initial surface pressure of 10 dyne/cm. In all cases, when peptides with the same number of residues are compared, the concentration needed to increase the surface pressure of the film by 1 dyne/cm was inversely related to its hydrophobicity. A reasonably good correlation was found to exist between the calculated free energy of transfer of a polypeptide from ethanol to water (a measure of its hydrophobicity) and its ability to increase the surface pressure of the DMPC film (a measure of the extent of its interaction with the neutral lipid monolayer).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28014/1/0000450.pd

Approches moléculaires du mode d'action des opioïdes (Etude structurale et fonctionnelle du récepteur humain des opioïdes de type mu surexprimé en cellules d'insecte)

La prise en charge et le soulagement de la douleur représentent une nécessité médicale majeure. La morphine demeure l'antalgique le plus puissant utilisé en clinique mais elle comporte des effets indésirables. Elle agit principalement sur le récepteur des opioïdes de type mu, un récepteur appartenant à la superfamille des récepteurs couplés aux protéines G. Comprendre les interactions ligand-récepteur et les modifications conformationnelles qui en résultent constitue une étape indispensable à l'interprétation de l'action des opioïdes au niveau moléculaire. De plus, la conception de molécules à effet thérapeutique basée sur la structure des récepteurs apparaît de plus en plus comme un outil précieux en chimie médicinale. Dans le cas des opioïdes, cette stratégie n'est pas appplicable puisque aucune structure cristallographique de ces récepteurs n'est disponible. Or, ces récepteurs sont naturellement très faiblement exprimés. Des systèmes de surexpression appropriés sont donc requis pour obtenir d'importantes quantités de protéine pure et active. Ce travail a débuté par les essais de solubilisation et purification du récepteur humain des opioïdes de type mu (hMOR) surexprimé dans les cellules Sf9 infectées par un baculovirus recombinant. Face aux difficultés rencontrées, un nouveau système d'expression inductible basé sur l'obtention de lignées stables dans des cellules Schneider S2 de Drosophila melanogaster a été mis en place. De manière innovante, le récepteur hMOR a été couplé à l'EGFP afin de permettre le suivi, la localisation, la quantification de manière directe et le développement d'un protocole de purification. Le récepteur produit a été caractérisé d'un point de vue biochimique et pharmacologique et sa purification a été entreprise. En parallèle, nous avons souhaité adapter la technique de mesure de la capacité de liaison du récepteur EGFPhMOR par transfert d'énergie de résonance de fluorescence avec différents ligands opioïdes rendus fluorescents.Pain management and treatment represent a major unmet medical challenge. Morphine remains the most potent painkiller used clinically, despite secondary side effects. It principally acts on the mu opioid receptor that belongs to the superfamily of the G-Protein-Coupled receptors. Interpretation of the opioid mode of action at the molecular level needs that we understand how the receptor and ligands interact with each other and what are the associated conformational changes. Moreover, the integration of structure-based methods, virtual screening, and combinatorial chemistry can provide the basis for more efficient drug design. In the case of opioids, this strategy is not applicable because these receptors are not yet amenable to structure based drug design due to the lack of three-dimensional structures. These receptors are endogenously poorly expressed so appropriate overexpression systems are required to obtain the large amounts needed for crystallization. This work began with solubilization and purification trials of the human mu opioid receptor (hMOR) overexpressed in baculovirus-infected Sf9 cells. Because difficulties we encountered, a new system of expression based on stable cell lines and on an inducible expression in Drosophila melanogaster Schneider S2 cells was developed. In an innovating way, the receptor was fused to the EGFP to follow, localize, directly quantify and develop a purification protocol. The produced receptor EGFPhMOR was biochemically and pharmacologically characterized and we began the purification process. In parallel, we wished to adapt the measurement of the binding capacity of the receptor based on the fluorescence resonance energy transfer between the EGFP fused to the receptor and opioid ligands wa made fluorescent.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Expression hétérologue de récepteurs couplés aux protéines G dans la levure Pichia pastoris (Une contribution au projet MePNet)

La famille des Récepteurs Couplés aux Protéines G (RCPG) est la plus importante famille de récepteurs transmembranaires responsables de la transduction de signaux extracellulaires. Cette famille protéique représente l'une des plus importantes catégories de cibles thérapeutiques puisqu'elles sont la cible de près de 60% des médicaments actuels (Howard et al. 2001, TIPS 22:132-140). Le travail présenté dans ce manuscrit est une contribution au projet de recherche européen MePNet (Membrane Protein Network) (Lundstrom K. 2004, Curr Opin Drug Dicscov Devel. 3:342-346). Le but de ce projet de biologie structurale consiste à exprimer une centaine de protéines membranaires de la famille des RCPG dans trois systèmes d'expression différents (Escherichia coli, Pichia pastoris et cellules de mammifères infectées par le Virus de la Forêt de Semliki - SFV). Ce travail de thèse a participé au développement de méthodologies adaptées aux contraintes du programme et permettant dans un premier temps le clonage des 100 gènes de RCPG sélectionnés pour le projet ainsi que la sélection des clones, l'expression et la caractérisation des récepteurs produits dans la levure P. pastoris. Ce travail s'est ensuite consacré à l'étude d'un panel de récepteur représentatif de la famille des RCPG et pour lesquels une étude plus approfondie a été réalisée afin d'optimiser les niveaux d'expression ainsi que les conditions de solubilisation et de purification.The superfamily of G protein-coupled receptors (GPCRs) mediate many important cellular signal transduction events. These membrane proteins are targets of 60% of the drugs nowadays used in therapeutics (Howard et al. 2001, TIPS 22:132-140). The work presented here was a contribution to an european research project MePNet (Membrane Protein Network) (Lundstrom K. 2004, Curr Opin Drug Dicscov Devel. 3:342-346). The goal of this structural biology project is to overexpress hundred GPCR in three different expression system (Escherichia coli, Pichia pastoris and mammalian cells infected by Semliki Forest Virus - SFV). This thesis project contributed to develop high throughput methods for the cloning of the 100 selected GPCR genes, but also for the selection, expression and the characterisation of the receptors produced in the yeast P. pastoris. Then, this work focused on the expression levels optimisation of a shorter panel of GPCRs and investigation of solubilisation and purification conditions.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Développement et optimisation des systèmes alphaviraux pour l'expression fonctionnelles des protéines membranaires

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Modèle d'étude de l'insertion des protéines membranaires

La colicine A produite par Citrobacter freundii et l'aérolysine produite par Aeromonas hydrophila sont deux protéines bien représentatives de classes très différentes de protéines formatrices de pores. La colicine A est une protéine formée uniquement d'hélices α qui possède une «épingle à cheveux» hydrophobe enfouie au sein de sa structure en milieu aqueux. C'est une «protéine membranaire dite inversée». L'aérolysine, en revanche, est formée essentiellement de feuillets β et ne possède pas de longue séquence hydrophob