Effect of cholesterol on the hydration properties of ester and ether lipid membrane interphases.

Fluorescence spectroscopy and Molecular Dynamics results show that cholesterol reduces water along the chains in ether lipids by changing the water distribution pattern between tightly and loosely bound water molecules. Water distribution was followed by emission spectra and generalized polarization of 6-dodecanoyl-2-dimethyl aminonaphthalene (Laurdan) inserted in 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14: 0 Diether PC) membranes. Molecular Dynamics simulations indicate that the action of cholesterol could be different in ether PC in comparison to ester PC. In addition, Cholesterol seems to act "per se" as an additional hydration center in ether lipids. Regardless of the phase state, cholesterol both in DMPC and 14:0 Diether PC vesicles, changed the distribution of water molecules decreasing the dipole relaxation of the lipid interphase generating an increase in the non-relaxable population. Above 10% Cholesterol/14:0 Diether PC ratio vesicles' interphase present an environment around Laurdan molecules similar to that corresponding to ester PC.

Interaction of chlorogenic acid with model lipid membranes and its influence on antiradical activity.

Chlorogenic acid (CGA) is a strong phenolic antioxidant with antibacterial properties composed by a caffeoyl ester of quinic acid. Although a number of benefits has been reported and related to interactions with the red blood cell membranes, details on its membrane action and how composition and membrane state may affect it, is not yet well defined. In this work, the interaction of CGA with lipid monolayers and bilayers composed by 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (16:0 diether PC) were studied at different surface pressures (π). The kinetics of interaction was found to be more rapid in DMPC than in the absence of carbonyl groups. Measurements by FTIR-ATR at different water activities confirm specific interactions of CGA with carbonyl and phosphate groups affecting water level along hydrocarbon region. The antioxidant activity of CGA in the presence of DMPC unilamellar vesicles, evidenced by the absorbance reduction of the radical cation ABTS•+, is significantly different with respect to aqueous solution. The influence of CGA on antiradical activity (ARA) with lipid membranes depending on the hydration state of the lipid interface is discussed.

Breakdown of classical paradigms in relation to membrane structure and functions.

Updates of the mosaic fluid membrane model implicitly sustain the paradigms that bilayers are closed systems conserving a state of fluidity and behaving as a dielectric slab. All of them are a consequence of disregarding water as part of the membrane structure and its essential role in the thermodynamics and kinetics of membrane response to bioeffectors. A correlation of the thermodynamic properties with the structural features of water makes possible to introduce the lipid membrane as a responsive structure due to the relaxation of water rearrangements in the kinetics of bioeffectors' interactions. This analysis concludes that the lipid membranes are open systems and, according to thermodynamic of irreversible formalism, bilayers and monolayers can be reasonable compared under controlled conditions. The inclusion of water in the complex structure makes feasible to reconsider the concept of dielectric slab and fluidity.

Water Behavior at the Phase Transition of Phospholipid Matrixes Assessed by FTIR Spectroscopy.

Lipid membranes are one of the most important biological matrixes in which biochemical processes take place. This particular lipid arrangement is driven by different water disposition interacting with it, which is related to different water states with different energy levels at the interphase. In our work, we report changes in water content and distinctive water states by Fourier transform infrared (FTIR) spectroscopy of this self-assembled matrix at different water contents and temperatures. To determine whether water properties at lipid interphases depend on the group of the lipid molecule at which it is bound the phase-transition temperature of 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC) was followed by the changes in frequency of the different groups of the lipids by attenuated total reflection (ATR)-FTIR spectroscopy at different humidities. A comparison of these two lipids enables us to put into relevance the contribution of the CO groups as a hydration site. These changes were compared with those occurring at the water band, and a value of the enthalpic change was evaluated from them. The -OH stretching in the liquid water IR spectrum is the principal region used to understand its molecular organization (4000-3000 cm-1). The strength of hydrogen bonding depends on the cooperative/anticooperative nature of the surrounding hydrogen bonds, with the strongest hydrogen bonds giving the lowest vibrational frequencies. Thus, we can use water as a mirror of the membrane state in this kind of biological systems. Different phospholipids associate water at particular modes according to their structures. This may produce modulation of packing and hydration suitable for the incorporation of amino acids, peptides, and enzymes.

Modulation of Interfacial Hydration by Carbonyl Groups in Lipid Membranes.

The lack of carbonyl groups and the presence of ether bonds give the lipid interphase a different water organization around the phosphate groups that affects the compressibility and electrical properties of lipid membranes. Generalized polarization of 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC) in correlation with Fourier transform infrared (FTIR) analysis indicates a higher level of polarizability of water molecules in the membrane phase around the phosphate groups both below and above Tm. This reorganization of water promotes a different response in compressibility and dipole moment of the interphase, which is related to different H bonding of water molecules with phosphates (PO) and carbonyl (CO) groups.

A new model for lipid monolayer and bilayers based on thermodynamics of irreversible processes.

Lipid monolayers are used as experimental model systems to study the physical chemical properties of biomembranes. With this purpose, surface pressure/area per molecule isotherms provide a way to obtain information on packing and compressibility properties of the lipids. These isotherms have been interpreted considering the monolayer as a two dimensional ideal or van der Waals gas without contact with the water phase. These modelistic approaches do not fit the experimental results. Based on Thermodynamics of Irreversible Processes (TIP), the expansion/compression process is interpreted in terms of coupled phenomena between area changes and water fluxes between a bidimensional solution of hydrated head groups in the monolayer and the bulk solution. The formalism obtained can reproduce satisfactorily the surface pressure/area per lipid isotherms of monolayer in different states and also can explain the area expansion and compression produced in particles enclosed by bilayers during osmotic fluxes. This novel approach gives relevance to the lipid-water interaction in restricted media near the membrane and provides a formalism to understand the thermodynamic and kinetic response of biointerphases to biological effectors.

Lipid selectivity in novel antimicrobial peptides: Implication on antimicrobial and hemolytic activity.

Antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. For an AMP to be considered as a therapeutic option, it must have not only potent antibacterial properties but also low hemolytic and cytotoxic activities [1]. Even though many studies have been conducted in order to correlate the antimicrobial activity with affinity toward model lipid membranes, the use of these membranes to explain cytotoxic effects (especially hemolysis) has been less explored. In this context, we studied lipid selectivity in two related novel AMPs, peptide 6 (P6) and peptide 6.2 (P6.2). Each peptide was designed from a previously reported AMP, and specific amino acid replacements were performed in an attempt to shift their hydrophobic moment or net charge. P6 showed no antimicrobial activity and high hemolytic activity, and P6.2 exhibited good antibacterial and low hemolytic activity. Using both peptides as a model we correlated the affinity toward membranes of different lipid composition and the antimicrobial and hemolytic activities. Our results from surface pressure and zeta potential assays showed that P6.2 exhibited a higher affinity and faster binding kinetic toward PG-containing membranes, while P6 showed this behavior for pure PC membranes. The final position and structure of P6.2 into the membrane showed an alpha-helix conversion, resulting in a parallel alignment with the Trps inserted into the membrane. On the other hand, the inability of P6 to adopt an amphipathic structure, plus its lower affinity toward PG-containing membranes seem to explain its poor antimicrobial activity. Regarding erythrocyte interactions, P6 showed the highest affinity toward erythrocyte membranes, resulting in an increased hemolytic activity. Overall, our data led us to conclude that affinity toward negatively charged lipids instead of zwitterionic ones seems to be a key factor that drives from hemolytic to antimicrobial activity.

The use of zeta potential as a tool to study phase transitions in binary phosphatidylcholines mixtures.

Temperature dependence of the zeta potential (ZP) is proposed as a tool to analyze the thermotropic behavior of unilamellar liposomes prepared from binary mixtures of phosphatidylcholines in the absence or presence of ions in aqueous suspensions. Since the lipid phase transition influences the surface potential of the liposome reflecting a sharp change in the ZP during the transition, it is proposed as a screening method for transition temperatures in complex systems, given its high sensitivity and small amount of sample required, that is, 70% less than that required in the use of conventional calorimeters. The sensitivity is also reflected in the pre-transition detection in the presence of ions. Plots of phase boundaries for these mixed-lipid vesicles were constructed by plotting the delimiting temperatures of both main phase transition and pre-transition vs. the lipid composition of the vesicle. Differential scanning calorimetry (DSC) studies, although subject to uncertainties in interpretation due to broad bands in lipid mixtures, allowed the validation of the temperature dependence of the ZP method for determining the phase transition and pre-transition temperatures. The system chosen was dipalmitoylphosphatidylcholine/dimyristoyl phosphatidylcholine (DMPC/DPPC), the most common combination in biological membranes. This work may be considered as a starting point for further research into more complex lipid mixtures with functional biological importance.

Interaction of Phenylalanine with DPPC Model Membranes: More Than a Hydrophobic Interaction.

The negative free energy previously reported is explained by the stabilization of a PC-Phe (phosphocholine-phenylalanine) complex in the presence of water shown by the decrease in the symmetric stretching frequency of the phosphate group of the lipid (PO2(-)). An entropic contribution due to the disruption of the water network around the phenyl and in the membrane defect may be invoked. The dipole potential decrease is explained by the orientation of the carboxylate opposing to the CO of the lipids with oxygen moiety toward the low hydrated hydrocarbon core. The symmetric bending frequency of NH3(+) group of Phe, decreases in 5.2 cm(-1) in relation to water congruent with zeta potential shift to positive values. The Phe to DPPC dissociation constant is Kd = 2.23 ± 0.09 mM, from which the free energy change is about -4.54 kcal/mol at 25 °C. This may be due to hydrophobic contributions and two hydrogen bonds.

Phenylalanine interaction with lipid monolayers at different pHs.

The influence of Phe on the surface pressure of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers at the air-water interface was studied at different initial surface pressures (26 and 40 mN/m) and two pHs (5.0 and 7.3) at constant temperature (20 °C). Changes produced by the aminoacid added to the subphase on the surface pressure and on the dipole potential of lipid monolayers were measured at a fixed area. Compressibility properties of the monolayers at different pHs were studied by (π-A) isotherms. The results suggest that Phe intercalates into a DPPC film at the air-water interface at pH 5 and forms a different arrangement at pH 7.3. The possible relevance of these results of the effect of Phe in physiological conditions is discussed.

Phenylalanine Blocks Defects Induced in Gel Lipid Membranes by Osmotic Stress.

We study the binding of phenylalanine (Phe) with dipalmitoylphosphocholine (DPPC) vesicles in gel (25 °C) and in liquid crystalline states (50 °C) and in gel large unilamellar vesicles (LUVs) subjected to osmotic dehydration with merocyanine (MC 540) as a fluorescent surface membrane marker. Phe does not produce significant changes in MC 540 monomer concentration in DPPC LUVs at 50 °C. In contrast, it significantly decreases the monomer adsorption in defects present in DPPC LUVs at 25 °C. When DPPC LUVs were subjected to hypertonic stress, dehydration caused more defects, and in this case phenylalanine is also able to block such defects.

Influence of temperature, anions and size distribution on the zeta potential of DMPC, DPPC and DMPE lipid vesicles.

The purpose of the work is to compare the influence of the multilamellarity, phase state, lipid head groups and ionic media on the origin of the surface potential of lipid membranes. With this aim, we present a new analysis of the zeta potential of multilamellar and unilamellar vesicles composed by phosphatidylcholines (PC) and phosphatidylethanolamines (PE) dispersed in water and ionic solutions of polarizable anions, at temperatures below and above the phase transition. In general, the adsorption of anions seems to explain the origin of the zeta potential in vesicles only above the transition temperature (Tc). In this case, the sign of the surface potential is ascribed to a partial orientation of head group moiety toward the aqueous phase. This is noticeable in PC head groups but not in PEs, due to the strong lateral interaction between PO and NH group in PE.

Functional role of water in membranes updated: A tribute to Träuble.

The classical view of a cell membrane is as a hydrophobic slab in which only nonpolar solutes can dissolve and permeate. However, water-soluble non-electrolytes such as glycerol, erythritol, urea and others can permeate lipid membranes in the liquid crystalline state. Moreover, recently polar amino acid's penetration has been explained by means of molecular dynamics in which appearance of water pockets is postulated. According to Träuble (1971), water diffuses across the lipid membranes by occupying holes formed in the lipid matrix due to fluctuations of the acyl chain trans-gauche isomers. These holes, named "kinks" have the molecular dimension of CH2 vacancies. The condensation of kinks may form aqueous spaces into which molecular species of the size of low molecular weight can dissolve. This molecular view can explain permeability properties considering that water may be distributed along the hydrocarbon chains in the lipid matrix. The purpose of this review is to consolidate the mechanism anticipated by Träuble by discussing recent data in literature that directly correlates the molecular state of methylene groups of the lipids with the state of water in each of them. In addition, the structural properties of water near the lipid residues can be related with the water activity triggering kink formation by changes in the head group conformation that induces the propagation along the acyl chains and hence to the diffusion of water.

Microthermodynamic interpretation of fluid states from FTIR measurements in lipid membranes: a Monte Carlo study.

Fourier transform infrared spectroscopy (FTIR) is usually employed to obtain transition temperatures of lipids and lipid mixtures and the effect on it of several effectors, such as cholesterol. However, no interpretation of the molecular information provided by the frequency shift to higher values observed at Tc is available. In this article, we demonstrate that data obtained by means of FTIR measurements contain information about the microscopic thermodynamics of the lipid-phase transition. By means of Monte Carlo simulation, we have been able to show that the frequency shift from low to high values can be taken as a two-state transition of molecular constituents in a lattice rearrangement. According to the model, at temperatures below Tc all of the groups are defined in the lowest-energy state defined by the lowest frequency value and therefore they are all connected in a gel lattice. Above Tc, some groups may reach different energy states depending on the restrictions imposed on the groups. Ideally, when all of the groups are able to reach the highest frequency, a fully "fluid" state is reached, which is a disordered state. If we take this hypothetical state as a reference, it is possible to show that the higher states become less accessible. The model is suitable for describing the effect of cholesterol, which is able to dump the phase transition and is congruent with previous data denoting that in the so-called fluid phase the first four to five methylene groups remain in the gel state even above Tc. The frequency value attained above Tc depends on the nature of the lipid acyl chain.

Structural and thermodynamic properties of water-membrane interphases: significance for peptide/membrane interactions.

Water appears as a common intermediary in the mechanisms of interaction of proteins and polypeptides with membranes of different lipid composition. In this review, how water modulates the interaction of peptides and proteins with lipid membranes is discussed by correlating the thermodynamic response and the structural changes of water at the membrane interphases. The thermodynamic properties of the lipid-protein interaction are governed by changes in the water activity of monolayers of different lipid composition according to the lateral surface pressure. In this context, different water populations can be characterized below and above the phase transition temperature in relation to the CH2 conformers' states in the acyl chains. According to water species present at the interphase, lipid membrane acts as a water state regulator, which determines the interfacial water domains in the surface. It is proposed that those domains are formed by the contact between lipids themselves and between lipids and the water phase, which are needed to trigger adsorption-insertion processes. The water domains are essential to maintain functional dynamical properties and are formed by water beyond the hydration shell of the lipid head groups. These confined water domains probably carries information in local units in relation to the lipid composition thus accounting for the link between lipidomics and aquaomics. The analysis of these results contributes to a new insight of the lipid bilayer as a non-autonomous, responsive (reactive) structure that correlates with the dynamical properties of a living system.

Antiradical activity of gallic acid included in lipid interphases.

Polyphenols are well known as antioxidant agents and by their effects on the hydration layers of lipid interphases. Among them, gallic acid and its derivatives are able to decrease the dipole potential and to act in water as a strong antioxidant. In this work we have studied both effects on lipid interphases in monolayers and bilayers of dimyristoylphosphatidylcholine. The results show that gallic acid (GA) increases the negative surface charges of large unilamellar vesicles (LUVs) and decreases the dipole potential of the lipid interphase. As a result, positively charged radical species such as ABTS(+) are able to penetrate the membrane forming an association with GA. These results allow discussing the antiradical activity (ARA) of GA at the membrane phase which may be taking place in water spaces between the lipids.

Surface and hysteresis properties of lipid interphases composed by head group substituted phosphatidylethanolamines.

This work analyzes the surface properties of PE-containing membranes modified at the head group region by the addition of methyl and ethyl residues at or near the amine group. These residues alter the lipid-lipid and lipid-water interactions by changes in the hydrogen bonding capability and the charge density of the amine group thus affecting the electrostatic interaction. The results obtained by measuring the dipole potential, the zeta potential, the area per lipid and the compressibility properties allow to conclude that the H-bonding capability prevails in the lipid-lipid interaction. The non polar groups attached to the C2-carbon of the ethanolamine chain introduces a steric hindrance against compression and increases the dipole potential. The analysis of areas suggests that lipids with methylated head groups have a much larger compressibility at expense of the elimination of hydration water, which is congruent with the broader extent of the hysteresis loop.

Connected and isolated CH2 populations in acyl chains and its relation to pockets of confined water in lipid membranes as observed by FTIR spectrometry.

Analysis of the band corresponding to the frequency of vibrational symmetric stretching mode of methylene groups in the lipid acyl chains and the bands of water below and above the phase transition of different lipids by Fourier transform infrared spectroscopy gives strong support to the formation of confined water pockets in between the lipid acyl chains. Our measures and analysis consolidate the mechanism early proposed by Traüble, in the sense that water is present in kinks formed by trans-gauche isomers along the hydrocarbon tails. The formation of these regions depends on the acyl lipid composition, which determines the presence of different populations of water species, characterized by its degree of H bond coordination in fluid saturated or unsaturated lipids. The free energy excess due to the reinforcement of the water structure along few water molecules in the adjacencies of exposed membrane residues near the phase transition is a reasonable base to explain the insertion and translocation of polar peptides and amino acid residues through the biomembrane on thermodynamic and structural grounds.

Water state and carbonyl distribution populations in confined regions of lipid bilayers observed by FTIR spectroscopy.

It has been suggested that water in confined regions presents different properties than bulk water, mainly because of the changes in water population species that may be induced by the adjacent walls of different polarities in terms of hydrogen bond formation. In this context, it would be expected that lipids in the gel and the fluid states should offer different templates for water organization. The presence of water pockets or defects in lipid bilayers has been proposed to explain the insertion of charged and polar peptides and amino acids in membranes. In this work, we provide direct evidence by means of FTIR spectroscopy that water band profiles are changed whether lipids are in the solid state, in the gel state after heating and cooling across the phase transition, or in the fluid state. The different bands found in each case were assigned to different H-bonded water populations in agreement with the exposure of carbonyl groups.

Role of guanidyl moiety in the insertion of arginine and Nalpha-benzoyl-L-argininate ethyl ester chloride in lipid membranes.

Guanidyl moieties of both arginine (Arg) and N(alpha)-benzoyl-L-argininate ethyl ester chloride (BAEE) are protonated in all environments studied, i.e., dry solid state, D(2)O solutions, and dry and hydrated lipids as suggested by DFT(B3LYP)/6-31+G(d,p) calculations. Arg and BAEE are able to insert in the lipid interphase of both DMPC and DOPC monolayers as revealed by the observed decrease in the membrane dipole potential they induce. The larger decrease in the dipole potential induced by BAEE, compared to Arg, can be explained partially by the higher affinity of the hydrophobic benzoyl and ethyl groups for the membrane phase, which allows an easier insertion of this molecule. FTIR studies indicate that the guanidyl moiety of Arg is with all probability facing the hydrophobic part of the lipids, whereas in BAEE this group is facing the water phase. Zeta potential measurements provide a direct evidence that Arg orients in the lipid interphase of phosphatidylcholine (PC) bilayers with the negative charged carboxylate group (-COO-) toward the aqueous phase.