Interaction of L-phenylalanine with carbonyl groups in mixed lipid membranes.

The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC.

Surface Characterization of Lipid Biomimetic Systems.

Zeta potential and dipole potential measures are direct operational methodologies to determine the adsorption, insertion and penetration of ions, amphipathic and neutral compounds into the membranes of cells and model systems. From these results, the contribution of charged and dipole groups can be deduced. However, although each method may give apparent affinity or binding constants, care should be taken to interpret them in terms of physical meaning because they are not independent properties. On the base of a recent model in which the lipid bilayer is considered as composed by two interphase regions at each side of the hydrocarbon core, this review describes how dipole potential and zeta potential are correlated due to water reorganization. From this analysis, considering that in a cell the interphase region the membrane extends to the cell interior or overlaps with the interphase region of another supramolecular structure, the correlation of dipole and electrostatic forces can be taken as responsible of the propagation of perturbations between membrane and cytoplasm and vice versa. Thus, this picture gives the membrane a responsive character in addition to that of a selective permeability barrier when integrated to a complex system.

Effect of cholesterol on the surface polarity and hydration of lipid interphases as measured by Laurdan fluorescence: New insights.

Comparison of the behavior of Laurdan in gel and in the liquid crystalline DPPC bilayers with that observed in chloroform and OctOH allow concluding that changes in the membrane lipid order cannot be ascribed to changes in viscosity of the local environment. Cholesterol acts as a spacer below the transition temperature of DPPC, promoting a disorder state in the acyl chain region. No evidence of water entrance has been detected with Laurdan up to 30% Cholesterol in DPPC in this condition. In contrast, Chol displaces to longer values the wavelength of Laurdan in membranes in the liquid crystalline state. This decrease in polarity occurs above 5% Chol and is directly related to the water extrusion produced by Chol. This effect is similar to that occurring in liquid crystalline membranes subjected to hypertonic stress. The behavior is comparable to that of Laurdan in OctOH at different water ratios below 5% Chol/DPPC. At higher ratios, other changes are evident.

Effect of 25-hydroxycholesterol in viral membrane fusion: Insights on HIV inhibition.

Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection. Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane. With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC.

Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) represent important self defense molecules in many organisms, including humans. These peptides have a broad spectrum of activities, killing or neutralizing many Gram-negative and Gram-positive bacteria. The emergence of multidrug resistant microbes has stimulated research on the development of alternative antibiotics. In the search for new antibiotics, cationic antimicrobial peptides (CAMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to lysis of microbial membranes and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing interest from clinical as well as basic research during the last decade. In this work, we studied the biophysical and microbiological characteristics of three new designed CAMPs. We modified a previously studied CAMP sequence, in order to increase or diminish the hydrophobic face, changing the position of two lysines or replacing three leucines, respectively. These mutations modified the hydrophobic moment of the resulting peptides and allowed us to study the importance of this parameter in the membrane interactions of the peptides. The structural properties of the peptides were also correlated with their membrane-disruptive abilities, antimicrobial activities and hemolysis of human red blood cells.

Water defects induced by expansion and electrical fields in DMPC and DMPE monolayers: contribution of hydration and confined water.

The values of capacitance of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) monolayers on Hg, derived from cyclic voltammetry studies indicate that when the lipids are near the phase transition temperature fractures are formed at a critical area beyond that corresponding to the hydration shell of the lipids in the liquid expanded state. Similar fractures are inferred to be formed when an electric field is applied at constant area, at a breaking potential which is a function of the lipid species. These voltage values denote that energy involved in the transition induced by the electrical field is much higher for DMPE than for DMPC at low areas. This can be explained by the higher intermolecular lateral interactions by H-bonds between the ethanolamine and phosphate groups. However, at larger areas, the energy values for DMPC are as high as for DMPE which is understood to be due to the higher hydration of phosphocholine head groups. This finding gives a new insight in relation to the dynamics of the lipid head groups at the membrane interphase region in terms of the states of water between the lipids. This is congruent with previous results evaluated with the well known ΔΠ vs. surface pressure plots in monolayers of the same lipids at air-water interfaces.

Methylation of ethanolamine groups in phosphoethanolamines is relevant for L-arginine insertion in lipid membranes.

The interaction of L-arginine with membranes composed by phospholipids with different degrees of methylation of the ethanolamine group was studied by means of surface and dipole potentials and surface pressure variations. The subsequent methylation of the amine head group appears to hinder the synergic response of the adsorption observed in phosphatidylethanolamine membranes. The kinetics of the binding process denotes that the methyl groups are relevant in regulating the specific interaction of the amino acid with the interface by hydrogen bonds. This response can be put in correlation with the function of signal transduction assigned previously to methyl lipids [F. Hirata and J. Axelrod, 1980] and appears to be relevant to understand the mechanism of insertion of arginine residues in peptides of biological interest.

Interaction of nicotinamide and picolinamide with phosphatidylcholine and phosphatidylethanolamine membranes: a combined approach using dipole potential measurements and quantum chemical calculations.

Interaction between the bioactive compounds nicotinamide and picolinamide and phospholipids (phosphatidylcholines and phosphatidylethanolamines) was investigated by a combined approach using dipole potential measurements and quantum chemical calculations. It is shown that nicotinamide and picolinamide interactions with phosphatidylcholines are of two main types: (i) specific interactions with the phosphate group of the lipid, for which H-bonding between NH(2) group of the substrate and the phosphate plays a dominant role, (ii) conjugated less specific weaker interactions involving both the phosphate and carbonyl groups of the head group, which propagate to the lipid alkyl chains and increase their conformational disorder. For phosphatidylethanolamines, picolinamide was found to decrease the dipole potential of the membrane in a similar way as for phosphatidylcholines, while nicotinamide is ineffective. These findings are correlated with the specific properties of phosphatidylethanolamines (reduced exposure of phosphate groups) and structural differences in the two substrates, in particular: different separation of the nitrogen atoms in the molecules, existence of a strong intramolecular hydrogen bond in picolinamide (NH...N ((ring))), which is absent in nicotinamide, and non-planarity of nicotinamide molecules, in contrast to picolinamide ones. Additional information on the lipid/substrate interactions was extracted from the analysis of the changes produced in the relevant vibrational frequencies of the lipid and substrate upon binding. The present study gives molecular support to the argument that changes of dipole potentials are due to effects on the constitutive dipolar PO and CO groups. In addition, it is also shown that according to the specific binding of the substrate to one or both of those, the conformational state of the acyl chains may be affected. These entropy effects may be in the origin of the well-known interdependence of the properties of one monolayer with respect to the other in bilayer membranes.

Características de las encapsulación de EGP, P64K y el conjugado de ambas proteínas en liposomas obtenidos mediante congelación-descongelación / Encapsulation characteristics of EGF, P64K and the conjugate of both proteins into liposomes obtained by freeze and thaw procedure

Se determinó la influencia de la composición fosfolipídica sobre la eficiencia de encapsulación de las siguientes proteínas recombinantes: factor de crecimiento epidérmico(EGF), P64K y el conjugado de ambas en liposomas obtenidos mediante el procedimiento tecnológico de congelación-descongelación. Se determinó además, la estabilidad de estos preparados durante el almacenamimento a 4 grados céntigrados. Se prepararon liposomas de dipalmitoi fosfatidilcolina, diesteaoril fosfatidilcolina y fosfatidilcolina de yema de huevo en todos los casos, con colesterol en proporción molar 1:1. Estos liposomas contenían al EGP y la P64K marcadas con I 125 o el conjugado marcado en la molécula del factor de crecimento. La capacidad de las vesículas liposomales para retener su contenido dependió no sólo de la composición fosfolipídica, en términos de su temperatura de transición, sino también de la naturaleza del soluto encapsulado. El EGP fue la entidad molecular que experimentó una mayor liberación de los liposomas al medio acuoso. Las vesículas de dipalmitoil fosfatidilcolina: colesterol resultaron las más estables en cuanto a su capacidad de retención de las proteínas recombinantes mientras que el conjugado se comportó como una entidad molecular diferente, al menos en relación a la composición fosfolipídica requerida para alcanzar una mayor estabilidad de los liposomas que lo contienen. La adicción de sacarosa, trealosa, maltosa o glucosa a las vesículas de fosfatidilcolina de yema de huevo: colesterol redujo notablemente la salida de EGP, independiente del tipo de azúcar empleado, en comparación con aquellas vesículas preparadas en ausencia de azúcares(AU)