Statistical models for experimental model membranes.

Biological membranes are constituted from lipid bilayers and proteins. Investigation of protein-membrane interaction, essential for biological function of cells, must rest upon solid knowledge of lipid bilayer behavior. Thus, extensive studies of an experimental model for membranes, lipid bilayers in water solution, have been undertaken in the last decades. These systems present structural, thermal and electrical properties which depend on temperature, ionic strength or concentration. In this talk, we shall discuss statistical models for lipid bilayers, as well as the relation between their properties and results for properties of lipid dispersions investigated by the laboratories supervised by Teresa Lamy (IF-USP) and Amando Ito (FFCL-USP).

Probing the interaction of brain fatty acid binding protein (B-FABP) with model membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that BFABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

EFFECT OF HEAD GROUP AND CURVATURE ON BINDING OF THE ANTIMICROBIAL PEPTIDE TRITRPTICIN TO LIPID MEMBRANES

In this work we examine the interaction between the 13-residue cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) and model membranes of variable lipid composition. The effect on peptide conformational properties was investigated by means of CD (circular dichroism) and fluorescence spectroscopies. Based on the hypothesis that the antibiotic acts through a mechanism involving toroidal pore formation, and taking into account that models of toroidal pores imply the formation of positive curvature, we used large unilamellar vesicles (LUV) to mimic the initial step of peptide-lipid interaction, when the peptide binds to the bilayer membrane, and micelles to mimic the topology of the pore itself, since these aggregates display positive curvature. In order to more faithfully assess the role of curvature, micelles were prepared with lysophospholipids containing (qualitatively and quantitatively) head groups identical to those of bilayer phospholipids. CD and fluorescence spectra showed that, while TRP3 binds to bilayers only when they carry negatively charged phospholipids. binding to micelles occurs irrespective of surface charge, indicating that electrostatic interactions play a less predominant role in the latter case. Moreover, the conformations acquired by the peptide were independent of lipid composition in both bilayers and micelles. However, the conformations were different in bilayers and in micelles, suggesting that curvature has an influence on the secondary structure acquired by the peptide. Fluorescence data pointed to an interfacial location of TRP3 in both types of aggregates. Nevertheless, experiments with a water soluble fluorescence quencher suggested that the tryptophan residues are more accessible to the quencher in micelles than in bilayers. Thus, we propose that bilayers and micelles can be used as models for the two steps of toroidal pore formation. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

What can light scattering spectroscopy do for membrane-active peptide studies

Highly charged peptides are important components of the immune system and belong to an important family of antibiotics. Although their therapeutic activity is known, most of the molecular level mechanisms are controversial. A wide variety of different approaches are usually applied to understand their mechanisms, but light scattering techniques are frequently overlooked. Yet, light scattering is a noninvasive technique that allows insights both on the peptide mechanism of action as well as on the development of new antibiotics. Dynamic light scattering (DLS) and static light scattering (SLS) are used to measure the aggregation process of lipid vesicles upon addition of peptides and molecular properties (shape, molecular weight). The high charge of these peptides allows electrostatic attraction toward charged lipid vesicles, which is studied by zeta potential (zeta-potential) measurements. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

Headgroup specificity for the interaction of the antimicrobial peptide tritrpticin with phospholipid Langmuir monolayers

We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE similar to DPPC << DPPA < DPPG). The stronger interaction with negatively charged phospholipids agrees with data for vesicles and planar lipid bilayers, and with AMPs greater activity against bacterial membranes versus mammalian cell membranes. Considerable expansion of negatively charged monolayers occurred at 10 and 30 mol% TRP3, especially at low surface pressure. Moreover, a difference was observed between PA and PG, demonstrating that the interaction, besides being modulated by electrostatic interactions, displays specificity with regard to headgroup, being more pronounced in the case of PG, present in large quantities in bacterial membranes. In previous studies, it was proposed that the peptide acts by a toroidal pore-like mechanism [1,2]. Considering the evidence from the literature that PG shows a propensity to form a positive curvature as do toroidal pores, the observation of TRP3's preference for the PG headgroup and the dramatic increase in area promoted by this interaction represent further support for the toroidal pore mechanism of action proposed for TRP3. (C) 2012 Elsevier B.V. All rights reserved.

Immunolocalization of bone morphogenetic protein 2 during the early healing events after guided bone regeneration

Objective. The objective of this study was to evaluate the immunolocalization of bone morphogenetic protein 2 (BMP-2) after autogenous block grafting covered or not with an e-PTFE membrane. Study Design. Forty-eight rats were divided into 2 groups, autogenous block graft (B) and autogenous block graft + e-PTFE membrane (MB), and were evaluated by immunohistochemistry at baseline and 3, 7, 14, 21, and 45 days. Results. The largest number of positive cells in the recipient bed was observed after 3 days in both groups. At the graft border, the largest number of positive cells was seen after 7 days in group B and after 14 days in group MB. The highest proportion of staining in the graft was observed after 3 days in group B and after 21 days in group MB. Conclusions. High proportions of stain were related to intense revascularization and osteogenesis. Except for the interface, BMP-2 staining occurred later in group MB than in group B in all structures analyzed. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;113:533-541)

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Tip indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.

Photo-activated phase separation in giant vesicles made from different lipid mixtures

Using giant unilamellar vesicles (GUVs) made from POPC. DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance. (C) 2011 Elsevier B.V. All rights reserved.

Interaction of gramicidin with DPPC/DODAB bilayer fragments

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids. (C) 2012 Elsevier B.V. All rights reserved.

Rotational diffusion membrane and fluorescence anisotropy.

Structural properties of model membranes, such as lipid vesicles, may be investigated through the addition of fluorescent probes. After incorporation, the fluorescent molecules are excited with linearly polarized light and the fluorescence emission is depolarized due to translational as well as rotational diffusion during the lifetime of the excited state. The monitoring of emitted light is undertaken through the technique of time-resolved fluorescence: the intensity of the emitted light informs on fluorescence decay times, and the decay of the components of the emitted light yield rotational correlation times which inform on the fluidity of the medium. The fluorescent molecule DPH, of uniaxial symmetry, is rather hydrophobic and has collinear transition and emission moments. It has been used frequently as a probe for the monitoring of the fluidity of the lipid bilayer along the phase transition of the chains. The interpretation of experimental data requires models for localization of fluorescent molecules as well as for possible restrictions on their movement. In this study, we develop calculations for two models for uniaxial diffusion of fluorescent molecules, such as DPH, suggested in several articles in the literature. A zeroth order test model consists of a free randomly rotating dipole in a homogeneous solution, and serves as the basis for the study of the diffusion of models in anisotropic media. In the second model, we consider random rotations of emitting dipoles distributed within cones with their axes perpendicular to the vesicle spherical geometry. In the third model, the dipole rotates in the plane of the of bilayer spherical geometry, within a movement that might occur between the monolayers forming the bilayer. For each of the models analysed, two methods are used by us in order to analyse the rotational diffusion: (I) solution of the corresponding rotational diffusion equation for a single molecule, taking into account the boundary conditions imposed by the models, for the probability of the fluorescent molecule to be found with a given configuration at time t. Considering the distribution of molecules in the geometry proposed, we obtain the analytical expression for the fluorescence anisotropy, except for the cone geometry, for which the solution is obtained numerically; (II) numerical simulations of a restricted rotational random walk in the two geometries corresponding to the two models. The latter method may be very useful in the cases of low-symmetry geometries or of composed geometries.