Lipid bilayer pre-transition as the beginning of the melting process

We investigate the bilayer pre-transition exhibited by some lipids at temperatures below their main phase transition, and which is generally associated to the formation of periodic ripples in the membrane. Experimentally we focus on the anionic lipid dipalmytoylphosphatidylglycerol (DPPG) at different ionic strengths, and on the neutral lipid dipalmytoylphosphatidylcholine (DPPC). From the analysis of differential scanning calorimetry traces of the two lipids we find that both pre- and main transitions are part of the same melting process. Electron spin resonance of spin labels and excitation generalized polarization of Laurdan reveal the coexistence of gel and fluid domains at temperatures between the pre- and main transitions of both lipids, reinforcing the first finding. Also, the melting process of DPPG at low ionic strength is found to be less cooperative than that of DPPC. From the theoretical side, we introduce a statistical model in which a next-nearest-neighbor competing interaction is added to the usual two-state model. For the first time, modulated phases (ordered and disordered lipids periodically aligned) emerge between the gel and fluid phases as a natural consequence of the competition between lipid-lipid interactions. (C) 2009 Elsevier B.V. All rights reserved.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

On the interaction of the anthraquinone barbaloin with negatively charged DMPG bilayers

Barbaloin is a bioactive glycosilated 1,8-dihydroxyanthraquinone present in several exudates from plants, Such as Aloe vera, which are used for cosmetic or food purposes. It has been shown that barbaloin interacts with DMPG (dimyristoylphosphatidylglycerol) model membranes, altering the bilayer structure (Alves, D. S.; Perez-Fons, L.; Estepa, A.; Micol, V. Biochem. Pharm. 2004, 68, 549). Considering that ESR (electron spin resonance) of spin labels is one of the best techniques to monitor structural properties at the molecular level, the alterations caused by the anthraquinone barbaloin on phospholipid bilayers will be discussed here via the ESR signal of phospholipid spin probes intercalated into the membranes. In DMPG at high ionic strength (10 mM Hepes pH 7.4 + 100 mM NaCl), a system that presents a gel-fluid transition around 23 degrees C, 20 mol % barbaloin turns the gel phase more rigid, does not alter much the fluid phase packing, but makes the lipid thermal transition less sharp. However, in a low-salt DMPG dispersion (10 mM Hepes pH 7.4 + 2 mM NaCl), which presents a rather complex gel-fluid thermal transition (Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19), barbaloin strongly affects bilayer structural properties, both in the gel and fluid phases, extending the transition region to much higher temperature values. The position of barbaloin in DMPG bilayers will be discussed on the basis of ESR results, in parallel with data from sample viscosity, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering).

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and H-2 NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, 2 H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S-mol) and dynamics (T-1) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a ""transient site"", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel. (C) 2007 Elsevier B.V. All rights reserved.

Molecular Dynamics Investigations of PRODAN in a DLPC Bilayer

Molecular dynamics computer simulations have been performed to identify preferred positions of the fluorescent probe PRODAN in a fully hydrated DLPC bilayer in the fluid phase. In addition to the intramolecular charge-transfer first vertical excited state, we considered different charge distributions for the electronic ground state of the PRODAN molecule by distinct atomic charge models corresponding to the probe molecule in vacuum as well as polarized in a weak and a strong dielectric solvent (cyclohexane and water). Independent on the charge distribution model of PRODAN, we observed a preferential orientation of this molecule in the bilayer with the dimethylamino group pointing toward the membrane's center and the carbonyl oxygen toward the membrane's interface. However, changing the charge distribution model of PRODAN, independent of its initial position in the equilibrated DLPC membrane, we observed different preferential positions. For the ground state representation without polarization and the in-cyclohexane polarization, the probe maintains its position close to the membrane's center. Considering the in-water polarization model, the probe approaches more of the polar headgroup region of the bilayer, with a strong structural correlation with the choline group, exposing its oxygen atom to water molecules. PRODAN's representation of the first vertical excited state with the in-water polarization also approaches the polar region of the membrane with the oxygen atom exposed to the bilayer's hydration shell. However, this model presents a stronger structural correlation with the phosphate groups than the ground state. Therefore, we conclude that the orientation of the PRODAN molecule inside the DLPC membrane is well-defined, but its position is very sensitive to the effect of the medium polarization included here by different models for the atomic charge distribution of the probe.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.

Hybrid materials from intermolecular associations between cationic lipid and polymers

Intermolecular associations between a cationic lipid and two model polymers were evaluated from preparation and characterization of hybrid thin films cast on silicon wafers. The novel materials were prepared by spin-coating of a chloroformic solution of lipid and polymer on silicon wafer. Polymers tested for miscibility with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) were polystyrene (PS) and poly(methyl methacrylate) (PMMA). The films thus obtained were characterized by ellipsometry, wettability, optical and atomic force microscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and activity against Escherichia coli. Whereas intermolecular ion-dipole interactions were available for the PMMA-DODAB interacting pair producing smooth PMMA-DODAB films, the absence of such interactions for PS-DODAB films caused lipid segregation, poor film stability (detachment from the silicon wafer) and large rugosity. In addition, the well-established but still remarkable antimicrobial DODAB properties were transferred to the novel hybrid PMMA/DODAB coating, which is demonstrated to be highly effective against E. coli.

Determinação do gradiente de pH na região interna a duas membranas em equilíbrio com eletrólito

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Photo-induced destruction of giant vesicles in methylene blue solutions

We study the photodecomposition of phospholipid bilayers in aqueous solutions of methylene blue. Observation of giant unilamellar vesicles under an optical microscope reveals a consistent pattern of membrane disruption as a function of methylene blue concentration and photon density for different substrates supporting the vesicles.

Active site mutants of human secreted Group IIA Phospholipase A(2) lacking hydrolytic activity retain their bactericidal effect

The Human Secreted Group IIA Phospholipase A(2) (hsPIA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K h5PLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 mu g/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca2+-independent damaging activity against Liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein (C) 2011 Elsevier Masson SAS. All rights reserved.

Coupling Between GABA(A)-R Ligand-Binding Activity and Membrane Organization in beta-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with beta-cyclodextrin (beta-CD). The mere pre-incubation (PI) at 37A degrees C accompanying the beta-CD treatment was an underlying source of perturbations increasing [H-3]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4A degrees C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the beta-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not beta-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with beta-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, beta-CD is not completely eliminated from the system through centrifugation washings. It was concluded that beta-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual beta-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5 +/- 1.8 angstrom, in contrast to only 2 +/- 1.5 angstrom usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na+ was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors. (C) 2011 Elsevier B.V. All rights reserved.

Membrane interactions of S100A12 (calgranulin C)

S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca2+, Zn2+ or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca2+ and Zn2+ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.

The contribution of surface residues to membrane binding and ligand transfer by the -tocopherol transfer protein ( -TTP)

Previous work has shown that the -tocopherol transfer protein ( -TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which -TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of -TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of -TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired -TTP-assisted secretion of -tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC (1)H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS. (C) 2011 Elsevier B.V. All rights reserved.