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.

Investigating the Impact of the Glycolipid Content on Aurein 1.2 Pores in Prokaryotic Model Bilayers: A Coarse-Grain Molecular Dynamics Simulation Study.

Aurein 1.2 is an antimicrobial peptide (AMP) with known lytic activity against bacterial membranes. Our previous studies revealed a differential action of aurein by both experimental and computational methods. This differential action was over membranes of two related probiotic strains, where the main difference between membranes was the number of glycolipids in the lipid composition. In this work, we aimed to investigate the interaction of aurein 1.2 with model bacterial membranes of varying glycolipid content. To this end, we performed extensive molecular dynamics simulations using the MARTINI coarse-grain force field and differential mixtures of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and monogalactosylglycerol (MG). We found a correlation between the presence of MG in PG/PE mixtures and the difficulty of aurein to stabilize pore structures, suggesting an AMP-resistance factor encoded in the lipid composition of the membrane. These findings may shed light on a possible mechanism of bacterial resistance to AMPs that is related to the glycolipid content of bacterial membranes.

Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes.

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (ß-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers. Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria. In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible. Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to ß defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.

Superficially active water in lipid membranes and its influence on the interaction of an aqueous soluble protease.

The purpose of this paper is to demonstrate that the interaction of an aqueous soluble enzyme with lipid membranes is influenced by the lipid composition of the interphase. The results show that the interaction of an aqueous soluble protease, Rennet from Mucor miehei, depends on the exposure of the carbonyl and phosphate groups at the membrane interphase. The changes produced by the protease on the surface pressure of monolayers of dimyristoylphosphatidylcholine (DMPC); dioleoylphosphatidylcholine (DOPC); diphytanoylphosphatidylcholine (DPhPC); dipalmitoylphosphatidylcholine (DPPC); di-O-tetradecylphosphatidyl-choline [D(ether)PC]; dimyristoylphosphatidylethanolamine (DMPE); di-O-tetradecyl-phosphatidylethanolamine [D(ether)PE] were measured at different initial surface pressures. The meaning of the DeltaPi vs. Pi curves was interpreted in the light of the concept of interphase given by Defay and Prigogine [R. Defay, I. Prigogine, Surface Tension and Adsorption, John Wiley & Sons, New York, 1966, pp. 273-277] considering the interphase as a bidimensional solution of polar head groups. With this approach, and based on reported evidences that carbonyls and phosphates are the main hydration sites of the lipid membranes, it is suggested that the mechanism of interaction of aqueous soluble protein involves water beyond the hydration shell. At high surface pressure, only water strongly bound to carbonyl and phosphate groups is present and the interaction is not occurring. In contrast, at low surface pressures, the protease-membrane interaction is a function of acyl chain for different polar groups. This is interpreted, as a consequence of the changes in the interfacial tension produced by the displacement of water confined between the hydrated head groups.

A coarse-grained approach to studying the interactions of the antimicrobial peptides aurein 1.2 and maculatin 1.1 with POPG/POPE lipid mixtures.

In the present work we investigated the differential interactions of the antimicrobial peptides (AMPs) aurein 1.2 and maculatin 1.1 with a bilayer composed of a mixture of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We carried out molecular dynamics (MD) simulations using a coarse-grained approach within the MARTINI force field. The POPE/POPG mixture was used as a simple model of a bacterial (prokaryotic cell) membrane. The results were compared with our previous findings for structures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a representative lipid of mammalian cells. We started the simulations of the peptide-lipid system from two different initial conditions: peptides in water and peptides inside the hydrophobic core of the membrane, employing a pre-assembled lipid bilayer in both cases. Our results show similarities and differences regarding the molecular behavior of the peptides in POPE/POPG in comparison to their behavior in a POPC membrane. For instance, aurein 1.2 molecules can adopt similar pore-like structures on both POPG/POPE and POPC membranes, but the peptides are found deeper in the hydrophobic core in the former. Maculatin 1.1 molecules, in turn, achieve very similar structures in both kinds of bilayers: they have a strong tendency to form clusters and induce curvature. Therefore, the results of this study provide insight into the mechanisms of action of these two peptides in membrane leakage, which allows organisms to protect themselves against potentially harmful bacteria. Graphical Abstract Aurein pore structure (green) in a lipid bilayer composed by POPE (blue) and POPG (red) mixture. It is possible to see water beads (light blue) inside the pore.

Clonidine complexation with hydroxypropyl-beta-cyclodextrin: From physico-chemical characterization to in vivo adjuvant effect in local anesthesia.

Clonidine (CND), an alpha-2-adrenergic agonist, is used as an adjuvant with local anesthetics. In this work, we describe the preparation and characterization of an inclusion complex of clonidine in hydroxypropyl-beta-cyclodextrin (HP-ß-CD), as revealed by experimental (UV-vis absorption, SEM, X-ray diffraction, DOSY- and ROESY-NMR) and theoretical (molecular dynamics) approaches. CND was found to bind to HP-ß-CD (Ka=20M(-1)) in 1:1 stoichiometry. X-ray diffractograms and SEM images provided evidence of inclusion complex formation, which was associated with changes in the diffraction patterns of the pure compounds. NMR experiments revealed changes in the chemical shift of H3HP-ß-CD hydrogens (Δ=0.026ppm) that were compatible with the insertion of CND in the hydrophobic cavity of the cyclodextrin. Molecular dynamics simulation with the three CND species that exist at pH 7.4 revealed the formation of intermolecular hydrogen bonds, especially for the neutral imino form of CND, which favored its insertion in the HP-ß-CD cavity. In vitro assays revealed that complexation retarded drug diffusion without changing the intrinsic toxicity of clonidine, while in vivo tests in rats showed enhanced sensory blockade after the administration of 0.15% CND, with the effect decreasing in the order: CND:HP-ß-CD+bupivacaine>CND+bupivacaine>bupivacaine>CND:HP-ß-CD>clonidine. The findings demonstrated the suitability of the complex for use as a drug delivery system for clinical use in antinociceptive procedures, in association with local anesthetics.

Effect of polar head groups on the activity of aspartyl protease adsorbed to lipid membranes.

The proteolytic activity of an aspartyl protease of Mucor miehei was correlated with the adsorption of the protease to lipid vesicles. It was observed that the presence of phosphatidylethanolamines (PE's) in the membrane increased the enzyme activity in a 20% in the gel phase and 10% in the fluid phase. The effects of protease on the surface pressure of monolayers composed by dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPE) were measured at constant temperature as a function of the surface pressure. At low surface pressures, the major changes were induced by protease on DOPC and DMPC monolayers. However, the effect were much lower when the monolayer was composed by DMPE. The low hydration and strong head-head interaction between the phosphates and the amine groups of adjacent PE's would result in an area per molecule much lower in PE than in phosphatidylcholine (PC) in concordance with the lower penetration in PE. Protease adsorption on PE membranes increases the proteolytic activity in which condition is less susceptible to inhibition by pepstatin. However, PC's do not alter the enzyme activity being the action of inhibitor unaffected.

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.