Fabiana Lairion

Structural and functional properties of hydration and confined water in membrane interfaces

The scope of the present review focuses on the interfacial properties of cell membranes that may establish a link between the membrane and the cytosolic components. We present evidences that the current view of the membrane as a barrier of permeability that contains an aqueous solution of macromolecules may be replaced by one in which the membrane plays a structural and functional role. Although this idea has been previously suggested, the present is the first systematic work that puts into relevance the relation water-membrane in terms of thermodynamic and structural properties of the interphases that cannot be ignored in the understanding of cell function. To pursue this aim, we introduce a new definition of interphase, in which the water is organized in different levels on the surface with different binding energies. Altogether determines the surface free energy necessary for the structural response to changes in the surrounding media. The physical chemical properties of this region are interpreted in terms of hydration water and confined water, which explain the interaction with proteins and could affect the modulation of enzyme activity. Information provided by several methodologies indicates that the organization of the hydration states is not restricted to the membrane plane albeit to a region extending into the cytoplasm, in which polar head groups play a relevant role. In addition, dynamic properties studied by cyclic voltammetry allow one to deduce the energetics of the conformational changes of the lipid head group in relation to the head-head interactions due to the presence of carbonyls and phosphates at the interphase. These groups are, apparently, surrounded by more than one layer of water molecules: a tightly bound shell, that mostly contributes to the dipole potential, and a second one that may be displaced by proteins and osmotic stress. Hydration water around carbonyl and phosphate groups may change by the presence of polyhydroxylated compounds or by changing the chemical groups esterified to the phosphates, mainly choline, ethanolamine or glycerol. Thus, surface membrane properties, such as the dipole potential and the surface pressure, are modulated by the water at the interphase region by changing the structure of the membrane components. An understanding of the properties of the structural water located at the hydration sites and the functional water confined around the polar head groups modulated by the hydrocarbon chains is helpful to interpret and analyze the consequences of water loss at the membranes of dehydrated cells. In this regard, a correlation between the effects of water activity on cell growth and the lipid composition is discussed in terms of the recovery of the cell volume and their viability. Critical analyses of the properties of water at the interface of lipid membranes merging from these results and others from the literature suggest that the interface links the membrane with the aqueous soluble proteins in a functional unit in which the cell may be considered as a complex structure stabilized by water rather than a water solution of macromolecules surrounded by a semi permeable barrier.

Structural and dynamical surface properties of phosphatidylethanolamine containing membranes.

The hydration of solid dimyristoylphosphatidylethanolamine (DMPE) produces a negligible shift in the asymmetric stretching frequency of the phosphate groups in contrast to dimyristoylphosphatidylcholine (DMPC). This suggests that the hydration of DMPE is not a consequence of the disruption of the solid lattice of the phosphate groups as occurs in DMPC. The strong lateral interactions between NH(3) and PO(2)(-) groups present in the solid PEs remain when the lipids are fully hydrated and seem to be a limiting factor for the hydration of the phosphate group hindering the reorientation of the polar heads. The lower mobility is reflected in a higher energy to translocate the phosphoethanolamine (P-N) dipoles in an electrical field. This energy is decreased in the presence of increasing ratios of PCs of saturated chains in phosphoethanolamine monolayer. The association of PC and PE in the membrane affecting the reorientation of the P-N groups is dependent of the chain-chain interaction. The dipole potentials of PCs and PEs mixtures show different behaviors according to the saturation of the acyl chain. This was correlated with the area in monolayers and the hydration of the P-N groups. In spite of the low hydration, DMPE is still able to adsorb fully hydrated proteins, although in a lower rate than DMPC at the same surface pressure. This indicates that PE interfaces possess an excess of surface free energy to drive protein interaction. The relation of this free energy with the low water content is discussed.

Role of guanidinium group in the insertion of l-arginine in DMPE and DMPC lipid interphases

l-Arginine (Arg) is a positively charged amino acid constituent of peptides and proteins, participating in diverse mechanisms of protein-membrane interaction. The effect of Arg on phosphatidylcholine (PC) membranes has been previously related to water structure changes and to the presence of water defects in the hydrocarbon region. However, no information is available with regard to phosphatidylethanolamine (PE), another important component of lipid membranes. For this reason, the aim of this study is to determine the effect of Arg on DMPE membranes and partially methylated PEs in comparison to DMPC. The adsorption of the amino acid onto the lipid membranes was followed by determining the changes in the surface potential as a function of the bulk amino acid concentrations. The effects of Arg on the surface properties were also measured by changes in the surface pressure and the dipole potential. The onset of the transition temperature was measured with a fluorophore anchored at the membrane interphase. The results provide a new insight on amino acid-PE interactions, which can be ascribed to specific perturbations in the head group region induced by the guanidinium residue.

Molecular Dynamics Study of the Interaction of Arginine with Phosphatidylcholine and Phosphatidylethanolamine Bilayers

In this work, the differential interaction of zwitterionic arginines with fully hydrated dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers was analyzed by molecular dynamics simulations. In both systems, arginine binds to lipids with the carboxylate moiety oriented toward the aqueous phase, in agreement with previous experimental determinations of ζ potential of DMPC and DMPE liposomes. The guanidinium groups are found at different depths within the bilayers indicating that some arginines are buried, especially in DMPE. We observe, in the DMPE system, that the strongest interaction occurs between the guanidinium group and the carbonyl oxygen of the lipid. In the case of DMPC membranes, the strongest interaction is found between the guanidinium groups of the arginines and the phosphate groups of the lipids. Unexpectedly, arginine zwitterions are stabilized through the creation of hydrogen bonds (HB), either with water or with polar groups of the lipids. The mechanisms of interaction seem to be different in both membranes. In DMPE bilayers, arginines insert by breaking the inner HB network of the polar head groups, consequently increasing the occupied area per lipid molecule. In the DMPC bilayers the arginines insert by replacing the already present water molecules within the membrane, without significant effects on the area per lipid.

Role of Guanidyl Moiety in the Insertion of Arginine and Nα-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.

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.

Oligoarginine vectors for intracellular delivery: role of arginine side-chain orientation in chain length-dependent destabilization of lipid membranes.

Arginine-rich peptides receive increased attention due to their capacity to cross different types of membranes and to transport cargo molecules inside cells. Even though peptide-induced destabilization has been investigated extensively, little is known about the peptide side-chain and backbone orientation with respect to the bilayer that may contribute to a molecular understanding of the peptide-induced membrane perturbations. The main objective of this work is to provide a detailed description of the orientation of arginine peptides in the lipid bilayer of PC and negatively charged PG liposomes using ATR-IR spectroscopy and molecular modeling, and to relate these orientational preferences to lipid bilayer destabilization. Molecular modeling showed that above the transition temperature arginine side-chains are preferentially solvent-directed at the PC/water interface whereas several arginine side-chains are pointing towards the PG hydrophobic core. IR dichroic spectra confirmed the orientation of the arginine side chains perpendicular to the lipid-water interface. IR spectra shows an randomly distributed backbone that seems essential to optimize interactions with the lipid membrane. The observed increase of permeation to a fluorescent dye is related to the peptide induced-formation of gauche bonds in the acyl chains. In the absence of hydrophobic residues, insertion of side-chains that favors phosphate/guanidium interaction is another mechanism of membrane permeabilization that has not been further analyzed so far.

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.