Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing.

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.

Preparação de nanocompósitos através do encapsulamento de polímeros condutores em 2H-MoS2 e 1T-TiS2

Nanocomposites obtained by the encapsulation of conducting polymers such as polyaniline and polydiphenylamine in 2H-MoS2 and 1T-TiS2 are synthesized and characterized by X-ray diffraction and infrared spectrophotometry. The synthesis consists in intercalating the layered compound with n-butyllithium and subsequent exfoliation in water and organic solvents. The nanocomposites are obtained by the adsorption of the polymers into the single-layers sulfides and posterior restacking. The X-ray diffraction measurements showed that the organic conducting polymers are encapsulated in mono and bilayers arrangement in a well-ordered fashion to produce single phase compounds.

Double-bilayer: a new phase formed by lysophospholipids and the corresponding fatty acid

The product of catalytic activity of the enzyme phospholipase A2, which resembles the core unit of animal toxins, on phospholipids is a 1:1 mixture of lysolipid and fatty acid. This mixture was studied by time-resolved simultaneous small- and wide angle x-ray diffraction over the temperature range from 23 to 53.5ºC. An unusually large lamellar structure was observed, with d = 11 nm, contradicting the complex functional dimer model between lysolipid and fatty acid. It can be explained by formation of a "double-bilayer", a new phase consisting of two different bilayers, one formed by lysophospholipid and other by fatty acid, bound together by head group interactions. Its strucutre was confirmed by simulations of the X-ray scattering pattern.

Membrane properties of structurally modified ceramides : effects on lipid lateral distribution and sphingomyelin-interactions in artificial bilayer membranes

Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology.

Studies on membrane properties of cholesterol and 3-beta modified sterol analogs

Cholesterol (Chol) is an important lipid in cellular membranes functioning both as a membrane fluidity regulator, permeability regulator and co-factor for some membrane proteins, e.g. G-protein coupled receptors. It also participates in the formation of signaling platforms and gives the membrane more mechanical strenght to prevent osmotic lysis of the cell. The sterol structure is very conserved and already minor structural modifications can completely abolish its membrane functions. The right interaction with adjacent lipids and the preference of certain lipid structures over others are also key factors in determining the membrane properties of cholesterol. Because of the many important properties of cholesterol it is of value to understand the forces and structural properties that govern the membrane behavior of this sterol. In this thesis we have used established fluorescence spectroscopy methods to study the membrane behavior of both cholesterol and some of its 3β-modified analogs. Using several fluorescent probes we have established how the acyl chain order of the two main lipid species, sphingomyelin (SM) and phosphatidylcholine (PC) affect sterol partitioning as well as characterized the membrane properties of 3β-aminocholesterol and cholesteryl phosphocholine. We concluded that cholesterol prefers SM over PC at equal acyl chain order, indicating that other structural properties besides the acyl chain order are important for sphingomyelin-sterol interactions. A positive charge at the 3β position only caused minor changes in the sterol membrane behavior compared to cholesterol. A large phosphocholine head group caused a disruption in membrane packing together with other membrane lipids with large head groups, but was also able to form stable fluid bilayers together with ceramide and cholesterol. The Ability of the large head group sterol to form bilayers together with ceramide was further explored in the last paper where cholesteryl phosphocholine/ceramide (Chol-PC/Cer) complexes were successfully used to transfer ceramide into cultured cells.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

X-ray diffraction of a gram-negative bacterial membrane mimetic

This thesis applies x-ray diffraction to measure he membrane structure of lipopolysaccharides and to develop a better model of a LPS bacterial melilbrane that can be used for biophysical research on antibiotics that attack cell membranes. \iVe ha'e Inodified the Physics department x-ray machine for use 3.'3 a thin film diffractometer, and have lesigned a new temperature and relative humidity controlled sample cell.\Ve tested the sample eel: by measuring the one-dimensional electron density profiles of bilayers of pope with 0%, 1%, 1G :VcJ, and 100% by weight lipo-polysaccharide from Pse'udo'lTwna aeTuginosa. Background VVe now know that traditional p,ntibiotics ,I,re losing their effectiveness against ever-evolving bacteria. This is because traditional antibiotic: work against specific targets within the bacterial cell, and with genetic mutations over time, themtibiotic no longer works. One possible solution are antimicrobial peptides. These are short proteins that are part of the immune systems of many animals, and some of them attack bacteria directly at the membrane of the cell, causing the bacterium to rupture and die. Since the membranes of most bacteria share common structural features, and these featuret, are unlikely to evolve very much, these peptides should effectively kill many types of bacteria wi Lhout much evolved resistance. But why do these peptides kill bacterial cel: '3 , but not the cells of the host animal? For gramnegative bacteria, the most likely reason is that t Ileir outer membrane is made of lipopolysaccharides (LPS), which is very different from an animal :;ell membrane. Up to now, what we knovv about how these peptides work was likely done with r !10spholipid models of animal cell membranes, and not with the more complex lipopolysa,echaricies, If we want to make better pepticies, ones that we can use to fight all types of infection, we need a more accurate molecular picture of how they \vork. This will hopefully be one step forward to the ( esign of better treatments for bacterial infections.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Études des interactions détergents/lipides dans les systèmes membranaires

Les liposomes sont des structures sphériques formés par l'auto-assemblage de molécules amphiphiles sous forme d'une bicouche. Cette bicouche sépare le volume intérieur du liposome du milieu extérieur, de la même manière que les membranes cellulaires. Les liposomes sont donc des modèles de membranes cellulaires et sont formulés pour étudier les processus biologiques qui font intervenir la membrane (transport de molécules à travers la membrane, effets des charges en surface, interactions entre la matrice lipidique et d'autres molécules, etc.). Parce qu'ils peuvent encapsuler une solution aqueuse en leur volume intérieur, ils sont aussi utilisés aujourd'hui comme nanovecteurs de principes actifs. Nous avons formulé des liposomes non-phospholipidiques riches en stérol que nous avons appelés stérosomes. Ces stérosomes sont composés d'environ 30 % d'amphiphiles monoalkylés et d'environ 70 % de stérols (cholestérol, Chol, et/ou sulfate de cholestérol, Schol). Quand certaines conditions sont respectées, ces mélanges sont capables de former une phase liquide ordonnée (Lo) pour donner, par extrusion, des vésicules unilamellaires. Certaines de ces nouvelles formulations ont été fonctionnalisées de manière à libérer leur contenu en réponse à un stimulus externe. En incorporant des acides gras dérivés de l’acide palmitique possédant différents pKa, nous avons pu contrôler le pH auquel la libération débute. Un modèle mathématique a été proposé afin de cerner les paramètres régissant leur comportement de libération. En incorporant un amphiphile sensible à la lumière (un dérivé de l’azobenzène), les liposomes formés semblent répondre à une radiation lumineuse. Pour ce système, il serait probablement nécessaire de tracer le diagramme de phase du mélange afin de contrôler la photo-libération de l’agent encapsulé. Nous avons aussi formulé des liposomes contenant un amphiphile cationique (le chlorure de cétylpyridinium). En tant que nanovecteurs, ces stérosomes montrent un potentiel intéressant pour la libération passive ou contrôlée de principes actifs. Pour ces systèmes, nous avons développé un modèle pour déterminer l’orientation des différentes molécules dans la bicouche. La formation de ces nouveaux systèmes a aussi apporté de nouvelles connaissances dans le domaine des interactions détergents-lipides. Aux nombreux effets du cholestérol (Chol) sur les systèmes biologiques, il faut ajouter maintenant que les stérols sont aussi capables de forcer les amphiphiles monoalkylés à former des bicouches. Cette nouvelle propriété peut avoir des répercussions sur notre compréhension du fonctionnement des systèmes biologiques. Enfin, les amphiphiles monoalkylés peuvent interagir avec la membrane et avoir des répercussions importantes sur son fonctionnement. Par exemple, l'effet antibactérien de détergents est supposé être dû à leur insertion dans la membrane. Cette insertion est régie par l'affinité existant entre le détergent et cette dernière. Dans ce cadre, nous avons voulu développer une nouvelle méthode permettant d'étudier ces affinités. Nous avons choisi la spectroscopie Raman exaltée de surface (SERS) pour sa sensibilité. Les hypothèses permettant de déterminer cette constante d’affinité se basent sur l’incapacité du détergent à exalter le signal SERS lorsque le détergent est inséré dans la membrane. Les résultats ont été comparés à ceux obtenus par titration calorimétrique isotherme (ITC). Les résultats ont montré des différences. Ces différences ont été discutées.

Étude des mécanismes moléculaires de formation des pores des toxines formeuses de pores par la spectroscopie de fluorescence

Les toxines formeuses de pore (PFTs) sont des protéines exogènes responsables d’un grand nombre de maladies infectieuses qui perméabilisent les membranes cellulaires de leur hôte. La formation des pores ou l’introduction d’une enzyme dans le cytoplasme peut entrainer l’apparition de symptômes de maladies connues (l’anthrax, le botulisme) et, dans le pire des cas, la mort. Les mécanismes d’infection et de destruction des cellules infectées sont bien caractérisés. Toutefois, l’aspect dynamique des changements de conformation durant le processus de perméabilisation reste à découvrir pour la majorité des toxines formeuses de pore. Le but de cette thèse est d’étudier les mécanismes d’oligomérisation des PFTs, ainsi que la formation des pores à la membrane lipidique grâce à la spectroscopie de fluorescence. Nous avons choisi la toxine Cry1Aa, un bio pesticide produit par le bacille de Thuringe et qui a été rigoureusement caractérisé, en tant que modèle d’étude. La topologie de la Cry1Aa à l’état actif et inactif a pu être résolue grâce à l’utilisation d’une technique de spectroscopie de fluorescence, le FRET ou transfert d’énergie par résonance entre un fluorophore greffé au domaine formeur de pore (D1) et un accepteur non fluorescent (le DPA ou dipicrylamine) localisé dans la membrane et qui bouge selon le potentiel membranaire. Le courant électrique, ainsi que la fluorescence provenant de la bicouche lipidique membranaire horizontale ont été enregistrés simultanément. De cette manière, nous avons pu localiser toutes les boucles reliant les hélices de D1 avant et après la formation des pores. Dans la forme inactive de la toxine, toutes ces boucles se trouvent du côté interne de la bicouche lipidique, mais dans sa forme active l’épingle α3-α4 traverse du côté externe, alors que toutes les autres hélices demeurent du côté interne. Ces résultats suggèrent que α3-α4 forment le pore. Nous avons découvert que la toxine change significativement de conformation une fois qu’elle se trouve dans la bicouche lipidique, et que la Cry1Aa attaque la membrane lipidique de l’extérieur, mais en formant le pore de l’intérieur. Dans le but de caractériser la distribution de toxines à chaque extrémité de la bicouche, nous avons utilisé une technique de double FRET avec deux accepteurs ayant des vitesses de translocation différentes (le DPA et l’oxonol) dans la membrane lipidique. De cette manière, nous avons déterminé que la toxine était présente des deux côtés de la bicouche lipidique durant le processus de perméabilisation. La dynamique d’oligomérisation de la toxine dans une bicouche lipidique sans récepteurs a été étudiée avec une technique permettant le compte des sauts de fluorescence après le photoblanchiment des fluorophore liés aux sous unités composant un oligomère présent dans la bicouche lipidique supportée. Nous avons confirmé de cette manière que la protéine formait ultimement des tétramères, et que cet état résultait de la diffusion des monomères de toxine dans la bicouche et de leur assemblage subséquent. Enfin nous avons voulu étudier le « gating » de la colicine Ia, provenant de la bactérie E.Coli, dans le but d’observer les mouvements que font deux positions supposées traverser la bicouche lipidique selon le voltage imposé aux bornes de la bicouche. Nos résultats préliminaires nous permettent d’observer un mouvement partiel (et non total) de ces positions, tel que le suggèrent les études de conductances du canal.