Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a HII growth in a lamellar Lc phase with a reduced degree of hydration. The HII phase occurs with the same repeat distance (dHII=5.4 nm) as the Lc phase and upon heating it expands considerably (deltad/deltaT approximately 0.22 nm/ degrees C). The dimensional stabilization of this HII phase coincides with the transition temperature of the Lc to Lalpha phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single HII phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase Lbeta with a repeat distance, dLbeta=6.5 nm, in co-existence with a HII phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid-lipid interactions through modulation of the membrane structure.     

Wetting and capillary condensation as means of protein organization in membranes.

Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes.     

Curvature effects on membrane-mediated interactions of inclusions

 We study the static, long-range interactions of inclusions embedded in lipid membranes. By using a two-dimensional model, we are able to determine explicitly the closed equilibrium shape of the membrane for any value of the distance between the inclusions; our results show that these shapes cannot be obtained by linearizing the equilibrium equations near a referential shape. Moreover, by computing the membrane-mediated force between the inclusions in given static conditions, we also detect the effects on the interactions due to the curvature and the closed geometry of the membrane. Received: 2 January 2001 / Revised version: 20 December 2001 / Published online: 26 June 2002     

The influence of vitamin K1 on the structure and phase behaviour of model membrane systems

Vitamin K1 is a component of the Photosystem I of plants which constitutes the major dietary form of vitamin K. The major function of this vitamin is to be cofactor of the microsomal gamma-glutamylcarboxylase. Recently, novel roles for this vitamin in the membrane have been postulated. To get insight into the influence of vitamin K1 on the phospholipid component of the membrane, we have studied the interaction between vitamin K1 and model membranes composed of dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylethanolamine (DEPE). We utilized high-sensitivity differential scanning calorimetry and small-angle X-ray diffraction techniques. Vitamin K1 affected the thermotropic properties of the phospholipids, broadened and shifted the transitions to lower temperatures, and produced the appearance of several peaks in the thermograms. The presence of the vitamin gave rise to the formation of vitamin-rich domains which were immiscible with the bulk phospholipid in both the gel and the liquid-crystalline phases. Vitamin K1 was unable to alter the lamellar organization of DMPC, but we found that it produced an increase in the interlamellar repeat spacing of DMPC at 10 degrees C. Interestingly, vitamin K1 promoted the formation of inverted hexagonal HII structures in the DEPE system. We discuss the possible implications that these vitamin K1-phospholipid interactions might have with respect to the biological function of the vitamin.     

Membrane-Mediated Aggregation of Curvature-Inducing Nematogens and?Membrane Tubulation

The shapes of cell membranes are largely regulated by membrane-associated, curvature-active proteins. Herein, we use a numerical model of the membrane, recently developed by us, with elongated membrane inclusions possessing spontaneous directional curvatures that could be different along, and perpendicular to, the membrane’s long axis. We show that, due to membrane-mediated interactions, these curvature-inducing membrane-nematogens can aggregate spontaneously, even at low concentrations, and change the local shape of the membrane. We demonstrate that for a large group of such inclusions, where the two spontaneous curvatures have equal sign, the tubular conformation and sometimes the sheet conformation of the membrane are the common equilibrium shapes. We elucidate the factors necessary for the formation of these protein lattices. Furthermore, the elastic properties of the tubes, such as their compressional stiffness and persistence length, are calculated. Finally, we discuss the possible role of nematic disclination in capping and branching of the tubular membranes.     

Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a H_II growth in a lamellar Lc phase with a reduced degree of hydration. The H_II phase occurs with the same repeat distance (d_HII=5.4 nm) as the Lc phase and upon heating it expands considerably (δd/δT≈0.22 nm/°C). The dimensional stabilization of this H_II phase coincides with the transition temperature of the Lc to L_α phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single H_II phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase L_β with a repeat distance, d_Lβ=6.5 nm, in co-existence with a H_II phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid–lipid interactions through modulation of the membrane structure.     

Effect of hydrophobic mismatch on phase behavior of lipid membranes

We investigate the competing effects of hydrophobic mismatch and chain stretching on the morphology and evolution of domains in lipid membranes via Monte Carlo techniques. We model the membrane as a binary mixture of particles that differ in their preferred lengths, with the shorter particles mimicking unsaturated nonraft lipids and the longer particles mimicking saturated raft lipids. We find that phase separation can be induced upon increasing either the ratio J/kappa of the hydrophobic surface tension J to the compressibility modulus kappa. J/kappa determines the decay length for thickness changes. When this decay length is larger than the system size the membrane remains mixed. Furthermore, increasing the thickness relaxation time can induce transient phase separation.     

Filter-feeding dabbling ducks (Anas spp.) can actively select particles by size

The relationship between bill morphology, function and prey use among filter-feeding dabbling ducks (Anas spp.) is poorly understood. In particular, it is not clear if interspecific differences in morphology affect the retention of prey to allow prey partitioning. The size of particles retained by ducks may be determined entirely by the distance between adjacent mandibular lamellae (interlamellar distance), possibly allowing interspecific partitioning of prey by size. Alternatively, articulation of the maxilla and mandible allows ducks to increase the distance between the maxillary and mandibular lamellae (lamellar separation) so that it exceeds their interlamellar distance, possibly allowing them to selectively expel unwanted particles larger than their interlamellar distance. In contrast, if interlamellar distance alone determines the size of particles ingested, particles larger than the interlamellar distance will always be retained because lamellar spacing is fixed. When large, preferred millet and wheat seeds were mixed with small, less preferred poppy seeds, all three ducks in this investigation ingested a greater proportion of the millet and wheat seeds than the poppy seeds, even though all three seed types were larger than the ducks' interlamellar distance. The proportion of millet and poppy seeds ingested when seeds were mixed differed from the proportion expected from foraging rates on unmixed prey, indicating the ducks actively avoided poppy seeds. These results are consistent with the lamellar separation hypothesis and clearly reject the singular role of interlamellar distance in prey retention. Because lamellar separation and water filtration rate are both determined by movement of the maxilla and mandible, there may be a trade-off between particle size selection and prey ingestion rate that allows interspecific partitioning of prey by size.     

X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers.

Phase separation in artificially stacked multilayers of isolated bovine retinal rod outer segment (ROS) membranes has been examined via x-ray diffraction and electron microscopy. Specimens were prepared by isopotential spin drying followed with partial hydration by equilibration against moist gas streams. Upon dehydration, the multilamellar membrane phase assumes a binary phase composition consisting of concentrated protein-containing lamellae interspersed with microdomains of hexagonally packed tubes of lipid in a HII configuration. The HII lattice is geometrically coupled to the lamellar phase with one set of hexagonal crystal planes co-planar to the local membrane lamellae. The hexagonal microdomains bear a striking resemblance to the "paracrystalline inclusions" observed in fast-frozen, intact frog ROS (Corless and Costello. 1981. Exp. Eye Res. 32:217). The lamellar lattice is characterized by an unusually small degree of disorder. Sharp lamellar diffraction with a 120 A unit cell is observed (at near total dehydration) to a resolution of 6 A. A model consistent with the data is that a multilamellar array of ROS disks is stable as long as the external disk surfaces are kept sufficiently far apart. If the distance between the membranes is allowed to shrink below a certain critical value, the disk lipids spontaneously convert to a nonbilayer phase. This suggests that the structure of the ROS is stabilized by an internal framework that acts to keep the disks apart from one another and from the plasmalemma. Thus, the necessity of avoiding phase separations may provide a rationale for the peculiar morphology of the ROS.     

The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes

We studied the interactions of the hypotensive drug, 2-hydroxyoleic acid (2OHOA), with model membranes using the techniques of DSC, 31P NMR and X-ray diffraction. We demonstrate that 2OHOA alters the thermotropic behaviour of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), thereby promoting the formation of hexagonal phases (H(II)), despite stabilizing the lamellar phase (Lalpha). The lattice parameters of lamellar and non-lamellar structures were not altered by the presence of 2OHOA. The molecular bases underlying the alterations in membrane structure provoked by 2OHOA were analysed by comparing the effects produced by 2OHOA with the closely related fatty acids (FAs), oleic acid (OA) and elaidic acid (EA). The capacity of C-18 FAs to induce H(II)-phase formation followed the order OA > 2OHOA > EA. Furthermore, while 2OHOA stabilized the Lalpha phase, OA destabilized it. The net negative charge of 2OHOA at physiological pH (approximately 7.4) influenced its effect on membrane structure. By analysing the molecular architecture of 2OHOA in DEPE monolayers, interactions between the carboxylate groups of 2OHOA and the amine groups of DEPE were observed, as well as between the 2-hydroxyl group of the FA and the carbonyl oxygen of the phospholipid acyl chain. These structural characteristics provoked an increase in the P-to-N and P-to-P distances of neighbouring phospholipid headgroups in the presence of 2OHOA, with respect to those observed with OA and EA. The higher headgroup area at the lipid-water interface in presence of 2OHOA could account for the differential effect of this drug on the phase behaviour of DEPE membranes.     

A differential scanning calorimetric and 31P NMR spectroscopic study of the effect of transmembrane alpha-helical peptides on the lamellar-reversed hexagonal phase transition of phosphatidylethanolamine model membranes

We have investigated the effects of the model alpha-helical transmembrane peptide Ac-K(2)L(24)K(2)-amide (L(24)) on the thermotropic phase behavior of aqueous dispersions of 1,2-dielaidoylphosphatidylethanolamine (DEPE) to understand better the interactions between lipid bilayers and the membrane-spanning segments of integral membrane proteins. We studied in particular the effect of L(24) and three derivatives thereof on the liquid-crystalline lamellar (L(alpha))-reversed hexagonal (H(II)) phase transition of DEPE model membranes by differential scanning calorimetry and (31)P nuclear magnetic resonance spectroscopy. We found that the incorporation of L(24) progressively decreases the temperature, enthalpy, and cooperativity of the L(alpha)-H(II) phase transition, as well as induces the formation of an inverted cubic phase, indicating that this transmembrane peptide promotes the formation of inverted nonlamellar phases, despite the fact that the hydrophobic length of this peptide exceeds the hydrophobic thickness of the host lipid bilayer. These characteristic effects are not altered by truncation of the side chains of the terminal lysine residues or by replacing each of the leucine residues at the end of the polyleucine core of L(24) with a tryptophan residue. Thus, the characteristic effects of these transmembrane peptides on DEPE thermotropic phase behavior are independent of their detailed chemical structure. Importantly, significantly shortening the polyleucine core of L(24) results in a smaller decrease in the L(alpha)-H(II) phase transition temperature of the DEPE matrix into which it is incorporated, and reducing the thickness of the host phosphatidylethanolamine bilayer results in a larger reduction in the L(alpha)-H(II) phase transition temperature. These results are not those predicted by hydrophobic mismatch considerations or reported in previous studies of other transmembrane alpha-helical peptides containing a core of an alternating sequence of leucine and alanine residues. We thus conclude that the hydrophobicity and conformational flexibility of transmembrane peptides can affect their propensity to induce the formation of inverted nonlamellar phases by mechanisms not primarily dependent on lipid-peptide hydrophobic mismatch.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.     

Short-range specific forces are able to induce hemifusion

Working with pure lipidic systems (giant unilamellar vesicles, 10-150 microm in diameter) as models for biological membranes, we have considered possible structures of the contact area of two adherent membranes by investigating the diffusion of fluorescent lipid analogues from one vesicle to another. Two bilayers in close contact can almost be seen as a lamellar structure in equilibrium. This is the usual configuration of two adherent vesicles, in which the interbilayer distance is estimated to be 3 nm. We have increased the attraction between the membranes by either adding depletion forces or by using a trick, inspired from the interaction between nucleic bases in nucleosides (herein adenosine and thymidine). The nucleosides were attached to the polar head of amphiphilic molecules that behave like phospholipids and were incorporated in the model membrane. The extra attraction between two membranes, resulting from base pairing, strongly decreased the interbilayer distance down to about 1 nm. This change of the water content induced lipid rearrangements, which could also be viewed in terms of a phase transition at low water content. These rearrangements were not observed in the case of depletion forces. We conclude that the introduction of an additional attractive force in the system modifies the equilibrium state, leading to a drastic change in the membrane behavior, which will tentatively be related to hemifusion.     

Conformational features of cell-bound drugs as detected by selective proton relaxation rate investigations

The nonselective and selective longitudinal relaxation rates were measured for procaine protons in the presence of model lipid membranes, biological membranes and whole cells. Unlike the nonselective relaxation rates, the selective rate was shown to be particularly sensitive in detecting binding interactions with macromolecular cell constituents. It was shown that the aromatic moiety of procaine is involved in binding the cell plasma membrane.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.     

Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.     

Transmembrane peptide-induced lipid sorting and mechanism of Lalpha-to-inverted phase transition using coarse-grain molecular dynamics

Molecular dynamics results are presented for a coarse-grain model of 1,2-di-n-alkanoyl-sn-glycero-3-phosphocholine, water, and a capped cylindrical model of a transmembrane peptide. We first demonstrate that different alkanoyl-length lipids are miscible in the liquid-disordered lamellar (Lalpha) phase. The transmembrane peptide is constructed of hydrophobic sites with hydrophilic caps. The hydrophobic length of the peptide is smaller than the hydrophobic thickness of a bilayer consisting of an equal mixture of long and short alkanoyl tail lipids. When incorporated into the membrane, a meniscus forms in the vicinity of the peptide and the surrounding area is enriched in the short lipid. The meniscus region draws water into it. In the regions that are depleted of water, the bilayers can fuse. The lipid headgroups then rearrange to solvate the newly formed water pores, resulting in an inverted phase. This mechanism appears to be a viable pathway for the experimentally observed Lalpha-to-inverse hexagonal (HII) peptide-induced phase transition.     

Kinetics of transport of hydrophobic ions through lipid membranes including diffusion polarization in the aqueous phase

Previous interpretations of the kinetics of transport of hydrophobic ions through membranes have been based on one of three limiting assumptions. Either diffusion in the aqueous phase was taken to be rapid, or ionic motion was constrained to the membrane or a steady state was presumed to be established within the membrane. We present a general treatment of the coupled diffusion process through both the aqueous phase and the membrane; our theory contains the previous results as limiting cases. It is applied to voltage jump-current relaxation experiments on black lipid membranes in the presence of dipicrylamine or sodium tetraphenylborate. We have attempted to establish the rate of desorption from the membrane. For the system phosphatidylserine/tetraphenylborate, the rate of desorption and the rate of translocation were found to be comparable.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.