- Email: [email protected]
Sphingomyelin - Cerebroside Exchange in Lipid Membrane Lateral Domain Segregation
Wednesday, February 15, 2017 we controlled the abundance of both PS and target molecules by fluorescence correlation spectroscopy. We conclude that the probability of a reactive moiety being oxidized is mainly governed by its distance to the SO source rather than by its chemical nature. Supported by Russian Scientific Fund N 14-1301373. 2577-Pos Board B184 Sphingomyelin - Cerebroside Exchange in Lipid Membrane Lateral Domain Segregation Emilio Gonza´lez-Ramı´rez, Alicia Alonso, Felix M. Goni. Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain. Galactocerebrosides, commonly referred to as cerebrosides (Ceb), are a family of glycosphingolipids, which contain a saccharide head group (galactose, glucose) covalently linked to a double-tailed ceramide via a glycosidic linkage through the primary hydroxyl. They can be found in neuronal tissues in the central nervous system, in the epithelial cells of the small intestine and colon and in the granular layer of the skin epidermis. Ceb, like sphingomyelins (SM), are found to be highly saturated in natural sources and both share a sphingoid backbone but with a different headgroup, a phosphorylcholine (SM) or a mono/oligosaccharide (Ceb). Their hydrogen bonding capability is due to the interaction between the saccharide group and the hydroxyl and amide groups of the sphingosine base. The aim of this study is to explore the effect of the exchange between sphingomyelin and cerebroside in the formation of segregated lateral domains. Natural origin lipids (ox brain) were used. The samples were analyzed by differential scanning calorimetry, confocal microscopy and atomic force microscopy in order to characterize lipid domains in supported planar bilayers and giant unilamellar vesicles. In general Ceb exhibit a much smaller tendency than SM to form bilayers at 37 C, either in the form of liposomes or as supported lipid bilayers. Their bilayer forming capacity increases notoriously when mixed with typical lamellar-phase lipids, e.g. dioleoyl phosphatidylcholine. Even under these conditions bilayer formation is extremely difficult at Ceb proportions above 25 mol%.
Membrane Active Peptides and Toxins II 2578-Pos Board B185 Calcium Tightly Regulates Disorder-To-Order Transitions Involved in the Secretion, Folding and Functions of the CyaA Toxin of Bordetella Pertussis, the Causative Agent of Whooping Cough Darragh P. O’Brien1, Sara E. Cannella1, Dominique Durand2, Ve´ronique Y. Ntsogo Engue´ne´1, Belen Hernandez3, Mahmoud Ghomi3, Orso Subrini1, Audrey Hessel1, Christian Malosse1, Ve´ronique Hourdel1, Patrice Vachette2, Julia Chamot-Rooke1, Se´bastien Brier1, Daniel Ladant1, Alexandre Chenal1. 1 Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2Institut de Biologie Inte´grative de la Cellule, UMR 9198, CNRS, ORSAY, France, 3 Groupe de Biophysique Mole´culaire, Universite´ Paris 13, Paris, France. The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Our results show that calcium is involved in several steps of this intoxication process. In the low calcium environment of the bacterial cytosol, the C-terminal, calcium-binding RTX domain of CyaA, RD, is an elongated, intrinsically disordered coil, appropriately sized for transport through the narrow secretion machinery. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favour the refolding of the upstream domains of the protein. Due to its hydrophobic character, CyaA is known for its propensity to aggregate into multimeric forms in the absence of a chaotropic agent in vitro. We have recently defined the experimental conditions required for CyaA folding, that is, calcium binding and molecular confinement (which may mimic the secretion channel constraints). Both parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric CyaA toxin exhibits efficient permeabilization in vitro and haemolytic activities in cellula; both activities are conserved even in a fully calcium-free environment. In contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane of the target cell, indicating that free calcium is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the adaptation of bacterial RTX toxins to the great variations in calcium concentrations encountered in the successive environments during the intoxication process.
523a
2579-Pos Board B186 Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using Artificial Bilayer Array with Fused Brush Border Membrane Vesicles from Colorado Potato Beetle Larvae Ekaterina Zaitseva1,2, Gerhard Baaken2, Victor M. Ruiz-Arroyo3, Inmaculada Garcı´a-Robles3, Camila Ochoa-Campuzano3, Galo A. Goig3, Amparo C. Martı´nez-Ramı´rez4, Carolina Rausell3, Jan Behrends1, Dolores Real3. 1 Institute of Physiology II, University of Freiburg, Freiburg, Germany, 2 Ionera Technologies, Freiburg, Germany, 3Department of Genetics, University of Valencia, Valencia, Spain, 4SCSIE, University of Valencia, Valencia, Spain. Bacillus thuringiensis Cry proteins are insecticidal toxins that bind to specific receptors on the brush border membrane of the susceptible insect midgut cells and induce cell lysis by inserting into the membrane and forming pores. Here we report a novel Cry3Aa toxin pore formation assay utilizingparallel lipid bilayer platform based on microelectrode cavity array (MECA) chip. The Cry 3Aa pore-forming activity has been tested and characterized in lipid bilayers fused with Brush Border Membrane Vesicles (BBMVs) as well as in receptor-free synthetic lipid membranes. First we developed protocols for efficient fusion of BBMVs isolated from the coleopteran pest Colorado potato beetle (CPB) (Leptinotarsa decemlineata) larvae with lipid membranes on the MECA chip. BBMVs fusion led to the incorporation of different types of membrane proteins and ion channels presented in the midgut epithelial membrane into the lipid bilayers. Addition of the Cry3Aa toxin to the bilayers fused with BBMVs from CPB larvae induced in approximately 70 % of the membranes pronounced changes in the membrane currents due to the toxin pore formation. The presence of receptor in the fused BBMVs allowed for reliable detection of Cry3Aa channel activity at toxin concentrations as low as 0.5 nM. In contrast to the native membrane preparations Cry3Aa pore forming ability in synthetic lipid bilayers was significantly lower. In receptor-free lipid membranes Cry3Aa pores were only observed at toxin concentrations above 14.5 nM. Most of the pores displayed a variable kinetic behavior with low open probability, complex activity patterns and multiple subconductance states. The present combination of the MECA-chip electrophysiology with native membrane preparations allows for rapid functional characterization of the insecticidal toxin pores and receptor-toxin interactions. 2580-Pos Board B187 Impact of Dendrimer Surface Chemistry on Anthrax Toxin Channel Blockage: A Single Molecule Study Goli Yamini, Ekaterina M. Nestorovich. Biology, The Catholic University of America, Washington, DC, USA. Multivalent ligands often possess an additive or cooperative affinity for a multivalent target which is significantly greater than that of a single functional group interacting with a monovalent target. This effect was recently investigated on a number of the anthrax toxin inhibitors assembled on cyclodextrin and dendrimer multivalent scaffolds. Multivalent binding can be strong despite individual bonds being relatively weak, yet from a drug design perspective, we typically strive to design multivalent compounds with high individual functional group affinity toward the respective binding site on a multivalent target. Keeping this requirement in mind, here we perform a single-channel/singlemolecule study to investigate kinetic parameters of the anthrax toxin PA63 channel blockage by generation 2 poly(amido amine) (PAMAM) dendrimers (G2) functionalized with different surface ligands, including G2-NH2, G2-OH, G2- succinamate, and G2-COONa. We found that the earlier reported difference in IC50 values of the G2-OH/PA63 and G2-NH2/PA63 binding reactions is determined by both on- and off-rates of the reversible dendrimer/channel binding reaction. In 1M KCl, we observed about 10 times decrease in both kon and toff with G2-OH compared to G2-NH2, whereas for both blockers kon and toff increased dramatically with the increase of transmembrane voltage. PAMAM dendrimers functionalized with negatively charged succinamate, but not carboxyl surface groups, were still reported to have some residual activity in inhibiting the anthrax toxin channels. The on-rate of the G2-succinamate binding was comparable to that of G2OH, however the off-time showed opposite voltage sensitivity. We also describe kinetics of the PA63 ion current modulation by two different types of the ‘‘imperfect’’ PAMAM dendrimers, - the mixed surface G2 75% OH/ 25% NH2 and G3-NH2 dendron. Both the blockers showed weaker voltage dependence compared to G2-NH2, however their KDs in 1M KCl were comparable.
Membrane Active Peptides and Toxins II 2578-Pos Board B185 Calcium Tightly Regulates Disorder-To-Order Transitions Involved in the Secretion, Folding and Functions of the CyaA Toxin of Bordetella Pertussis, the Causative Agent of Whooping Cough Darragh P. O’Brien1, Sara E. Cannella1, Dominique Durand2, Ve´ronique Y. Ntsogo Engue´ne´1, Belen Hernandez3, Mahmoud Ghomi3, Orso Subrini1, Audrey Hessel1, Christian Malosse1, Ve´ronique Hourdel1, Patrice Vachette2, Julia Chamot-Rooke1, Se´bastien Brier1, Daniel Ladant1, Alexandre Chenal1. 1 Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2Institut de Biologie Inte´grative de la Cellule, UMR 9198, CNRS, ORSAY, France, 3 Groupe de Biophysique Mole´culaire, Universite´ Paris 13, Paris, France. The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Our results show that calcium is involved in several steps of this intoxication process. In the low calcium environment of the bacterial cytosol, the C-terminal, calcium-binding RTX domain of CyaA, RD, is an elongated, intrinsically disordered coil, appropriately sized for transport through the narrow secretion machinery. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favour the refolding of the upstream domains of the protein. Due to its hydrophobic character, CyaA is known for its propensity to aggregate into multimeric forms in the absence of a chaotropic agent in vitro. We have recently defined the experimental conditions required for CyaA folding, that is, calcium binding and molecular confinement (which may mimic the secretion channel constraints). Both parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric CyaA toxin exhibits efficient permeabilization in vitro and haemolytic activities in cellula; both activities are conserved even in a fully calcium-free environment. In contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane of the target cell, indicating that free calcium is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the adaptation of bacterial RTX toxins to the great variations in calcium concentrations encountered in the successive environments during the intoxication process.
523a
2579-Pos Board B186 Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using Artificial Bilayer Array with Fused Brush Border Membrane Vesicles from Colorado Potato Beetle Larvae Ekaterina Zaitseva1,2, Gerhard Baaken2, Victor M. Ruiz-Arroyo3, Inmaculada Garcı´a-Robles3, Camila Ochoa-Campuzano3, Galo A. Goig3, Amparo C. Martı´nez-Ramı´rez4, Carolina Rausell3, Jan Behrends1, Dolores Real3. 1 Institute of Physiology II, University of Freiburg, Freiburg, Germany, 2 Ionera Technologies, Freiburg, Germany, 3Department of Genetics, University of Valencia, Valencia, Spain, 4SCSIE, University of Valencia, Valencia, Spain. Bacillus thuringiensis Cry proteins are insecticidal toxins that bind to specific receptors on the brush border membrane of the susceptible insect midgut cells and induce cell lysis by inserting into the membrane and forming pores. Here we report a novel Cry3Aa toxin pore formation assay utilizingparallel lipid bilayer platform based on microelectrode cavity array (MECA) chip. The Cry 3Aa pore-forming activity has been tested and characterized in lipid bilayers fused with Brush Border Membrane Vesicles (BBMVs) as well as in receptor-free synthetic lipid membranes. First we developed protocols for efficient fusion of BBMVs isolated from the coleopteran pest Colorado potato beetle (CPB) (Leptinotarsa decemlineata) larvae with lipid membranes on the MECA chip. BBMVs fusion led to the incorporation of different types of membrane proteins and ion channels presented in the midgut epithelial membrane into the lipid bilayers. Addition of the Cry3Aa toxin to the bilayers fused with BBMVs from CPB larvae induced in approximately 70 % of the membranes pronounced changes in the membrane currents due to the toxin pore formation. The presence of receptor in the fused BBMVs allowed for reliable detection of Cry3Aa channel activity at toxin concentrations as low as 0.5 nM. In contrast to the native membrane preparations Cry3Aa pore forming ability in synthetic lipid bilayers was significantly lower. In receptor-free lipid membranes Cry3Aa pores were only observed at toxin concentrations above 14.5 nM. Most of the pores displayed a variable kinetic behavior with low open probability, complex activity patterns and multiple subconductance states. The present combination of the MECA-chip electrophysiology with native membrane preparations allows for rapid functional characterization of the insecticidal toxin pores and receptor-toxin interactions. 2580-Pos Board B187 Impact of Dendrimer Surface Chemistry on Anthrax Toxin Channel Blockage: A Single Molecule Study Goli Yamini, Ekaterina M. Nestorovich. Biology, The Catholic University of America, Washington, DC, USA. Multivalent ligands often possess an additive or cooperative affinity for a multivalent target which is significantly greater than that of a single functional group interacting with a monovalent target. This effect was recently investigated on a number of the anthrax toxin inhibitors assembled on cyclodextrin and dendrimer multivalent scaffolds. Multivalent binding can be strong despite individual bonds being relatively weak, yet from a drug design perspective, we typically strive to design multivalent compounds with high individual functional group affinity toward the respective binding site on a multivalent target. Keeping this requirement in mind, here we perform a single-channel/singlemolecule study to investigate kinetic parameters of the anthrax toxin PA63 channel blockage by generation 2 poly(amido amine) (PAMAM) dendrimers (G2) functionalized with different surface ligands, including G2-NH2, G2-OH, G2- succinamate, and G2-COONa. We found that the earlier reported difference in IC50 values of the G2-OH/PA63 and G2-NH2/PA63 binding reactions is determined by both on- and off-rates of the reversible dendrimer/channel binding reaction. In 1M KCl, we observed about 10 times decrease in both kon and toff with G2-OH compared to G2-NH2, whereas for both blockers kon and toff increased dramatically with the increase of transmembrane voltage. PAMAM dendrimers functionalized with negatively charged succinamate, but not carboxyl surface groups, were still reported to have some residual activity in inhibiting the anthrax toxin channels. The on-rate of the G2-succinamate binding was comparable to that of G2OH, however the off-time showed opposite voltage sensitivity. We also describe kinetics of the PA63 ion current modulation by two different types of the ‘‘imperfect’’ PAMAM dendrimers, - the mixed surface G2 75% OH/ 25% NH2 and G3-NH2 dendron. Both the blockers showed weaker voltage dependence compared to G2-NH2, however their KDs in 1M KCl were comparable.