On the transport noise of hydrophobic ions in lipid bilayer membranes

The effect of transit time on the electrical transport noise of a closed one-barrier model at equilibrium as proposed by Kolb and Läuger [6] is studied using the master-equation approach. A transit time is the time for an ion to cross the energy barrier (membrane interior) when the energy of the ion reaches the barrier height. Both the time correlation function and the noise power spectrum are obtained as functions of the transit time of the ions. Possible effects of transit time on the time correlation function of transport of dipicrylamine ions in lipid bilayers as reported by Bruner and Hall [13] and on the noise power spectrum as reported by Kolb and Läuger [6] are discussed.     

Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ². From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.     

A study of Li+-selective permeation through lipid bilayer membranes mediated by a new ionophore (AS701)

The neutral, noncyclic, imide and ether containing ionophore AS701, has been developed as Li⁺-selective molecule, to be used potentially as an aid in the Li⁺-therapy of manic-depressive illness. The present report is a characterization of this molecule in neutral lipid bilayer membranes. This ionophore was found to the bilayers Li⁺-selective, acting as a selective carrier of monovalent cations. In addition, this molecule was found to be capable of acting as a selective carrier of monovalent anions. For both types of ions, the rate-limitting step in the process of permeation was found to be the diffusion of the carrier-ion complex through the membrane. The membrane-permeating species were found to be 2 : 1 carrier-ion complexes, carrying either a monovalent cation or a monovalent anion. The selectivity sequences among the ions studied being: Li⁺(1) > ClO₄^?(0.7) > Na⁺(0.07) > K⁺(0.016) > Rb⁺(0.0095) > Cs⁺(0.0083) > Cl^?(0.001). Mg²⁺ and SO₄^2? were found to be impermeant (under present experimental conditions). This sequence shows that the AS701 molecule has low selectivity for ions present in biological media, among those studied (i.e. Na⁺, K⁺, Mg²⁺, Cl^2? and SO₄^2?). This indicates that these ions will not interfere in the Li⁺ permeability induced by this carrier in vivo, and that the carrier will not interfere in the normal transport processes of these ions.     

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.     

Aggregation of chlorophylls in monolayers. V. The effect of water on chlorophyll a and chlorophyll b in mono and multilayer arrays

The nature of the interactions between water molecules and monolayers and multilayers of chlorophyll a (Chl a), and monolayers and multilayers of Chl b. obtained by the Langmuir-Blodgett technique, is examined by infrared spectroscopy. Following deposition of the monolayer or multilayer of Chl a or Chl b onto a plate, repetitive scans showed some modifications in the infrared spectra which are interpreted as a reorganization of the molecules as some water molecules leave the array. Drying the sample further modifies the spectra, which indicates the departure of more tenacious water molecules. Putting the sample in a moist atmosphere does not restore the original spectrum. This is an indication of a nonreversibie reorganization in the chlorophyll array. The spectra of the monolayer of chlorophyll are much more complicated than those of the multilayer, owing to the nonintegrating effect of the monolayer, which reveals the perturbing effect of the different dielectric milieux on each functional group. On the basis of the analysis of the spectra and the information gathered from the surface pressure isotherms, a model is proposed for the monolayer arrangement at the air/water interface which implies two set of dimers of water per molecule of chlorophyll. One pair of dimers constitutes the water of the first kind and is composed of vapor-like dimers. This kind of water is situated between the porphyrin planes of chlorophyll molecules and is easily removed from the monolayer. The second pair of dimers is composed of water of the second and third kinds situated between the Mg atom of the chlorophyll molecules and the water of the subphase. The second kind of water is closest to the Mg atom and is the most difficult one to remove. The third kind of water is closest to the surface and its mobility is intermediate between that of water of the first kind and that of water of the second kind. Comparing the infrared spectra of a freshly prepared monolayers of Chl a with the resonance Raman spectra of intact chloroplasts (M. Lutz, Biochim. Biophys. Acta 460 (1977) 408), we notice great similarities. This is an indication that the model we propose for the monolayer of Chl a could play an important role in the chloroplast.     

A simple theory of peptide interactions on a membrane surface: excluded volume and entropic order

A simple theory of the interactions of peptides bound onto a lipid membrane is developed, modeling the peptides as rods on a surface. At low peptide surface-concentration, excluded volume dominates the peptide-peptide interactions and the orientation of the peptides is random, resulting in an isotropic configuration. However, at high peptide density on the membrane, the peptides become orientationally ordered, resulting in an anisotropic configuration. This effect is entirely entropic in origin, and simply reflects the fact that peptides can be exchanged more easily on the surface if they are equally aligned, resulting in a larger number of possible configurations. In three dimensions, this phenomenon corresponds to the well-known isotropic-nematic phase transition. In two dimensions, the problem is not as well understood. The theoretical treatment presented here yields a simple, manageable expression which can be compared with experimental data. Two-dimensional ordering results in an increase in the apparent binding constant of peptides to membranes at high concentration of peptides relative to what is expected from the effect of excluded volume alone. The possible implications of side-by-side alignment for several biological processes, such as peptide translocation across membranes and plaque formation in Alzheimer's disease, are discussed.     

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes

Molecules analogous to biological and synthetic lipids have been prepared with conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10³-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.     

On the mechanism of SPP-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane

Intramembrane-cleaving proteases are members of a novel type of enzyme that hydrolyse substrate proteins within transmembrane regions. The presently known proteases that catalyse such cleavage reactions are membrane proteins of high hydrophobicity and multiple predicted transmembrane regions. A key feature is the positioning of active site residues in hydrophobic segments implying that the catalytic centre is assembled within the plane of the membrane. Nevertheless, all these proteases appear to utilise catalytic mechanisms similar to classic proteases that expose their active site domains in aqueous compartments. In the present review, we will address the mechanism of intramembrane proteolysis on the example of the signal peptide peptidase, and discuss how enzyme-catalysed hydrolysis of peptide bonds within the plane of a cellular membrane might occur.     

Outer membrane proteins: comparing X-ray and NMR structures by MD simulations in lipid bilayers

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 × 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the ‘quality’ of the starting structure seems mainly to influence the simulation stability of the transmembrane β-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal α-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a ∼10 ns timescale.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

A new method for membrane reconstitution: Fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).     

Gap junction channels reconstituted in two closely apposed lipid bilayers

Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.     

Lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers

A model recently used to study lipid-protein interactions in one-component lipid bilayers (Sperotto and Mouritsen, 1991 a, b) has been extended in order to include two different lipid species characterized by different acyl-chain lengths. The model, which is a statistical mechanical lattice model, assumes that hydrophobic matching between lipid-bilayer hydrophobic thickness and hydrophobic length of the integral protein is an important aspect of the interactions. By means of Monte Carlo simulation techniques, the lateral distribution of the two lipid species near the hydrophobic protein-lipid interface in the fluid phase of the bilayer has been derived. The results indicate that there is a very structured and heterogeneous distribution of the two lipid species near the protein and that the protein-lipid interface is enriched in one of the lipid species. Out of equilibrium, the concentration profiles of the two lipid species away from the protein interface are found to develop a long-range oscillatory behavior. Such dynamic membrane heterogeneity may be of relevance for determining the physical factors involved in lipid specificity of protein function.     

Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4'-dimethylaminochalcone

The dynamic behavior of polar molecules in egg phosphatidylcholine (PC) bilayers has been studied using a membrane fluorescent probe, 4'-dimethylaminochalcone (DMAC). Time and spectrally resolved fluorescence spectroscopy of DMAC incorporated in PC liposomes, as compared to studies of the probe in organic solvents, shows the existence of two independent populations, associated with different extent and speed of dipolar solvent relaxation. The first DMAC population represents approximately 69% of the fluorescence-emitting molecules, has a short fluorescence decay time (0.32 ns) and undergoes Stokes shift of 80 nm. The remaining 31% fraction of DMAC molecules has a decay time of 0.74 ns and undergoes a high (106 nm) Stokes shift. A fraction of the shift, ca. 24 nm for the first and 46 nm for the second population, is attributed to the fast (<0.1 ns) rotational relaxation of nearby dipolar molecules, which might be water. This two-state model accounts well for the detailed fluorescence properties of DMAC in egg PC, i.e. its broadened steady-state spectrum, its average fluorescence quantum yield and its complex wavelength-dependent fluorescence decays.     

The synthesis and use of thionphospholipids in 31P-NMR studies of lipid polymorphism

(1) Dipalmitoyl- and dioleoylthionphosphatidylcholine, which are phosphatidylcholine analogues in which the double bonded oxygen of the phosphate group is replaced by a sulfur atom, have been synthesized in 50–60% yields by condensation of diacylglycerol with phosphorus thionchloride in the presence of choline toluene-sulfonate. Dioleoylthionphosphatidylethanolamine has been prepared by the phospholipase D-catalyzed base exchange reaction. (2) Freeze-fracturing of aqueous dispersions of the thionphospholipids reveals that the thionphosphatidylcholines are organized in extended bilayers whereas dioleoylthionphosphatidylethanolamine above 0°C forms the hexagonal H_II phase similar to dioleoylphosphatidylethanolamine. The gel → liquid crystalline phase transition of the dipalmitoylthionphosphatidylcholine occurs at 44°C which is only slightly higher than the transition temperature of dipalmitoylphosphatidylcholine which together with other data demonstrates that the thionphospholipids closely resemble the natural phospholipids in physicochemical behaviour. (3) Proton decoupled ³¹P-NMR spectra of aqueous dispersions of thionphosphatidylcholines have the characteristic asymmetrical line-shape with a low-field shoulder and a high-field peak typical of phospholipids organized in extended bilayers in which the phosphate group can undergo fast axial rotation. The ³¹P-NMR spectrum of the thionphosphatidylethanolamine in the hexagonal H_II phase has a line-shape with a reversed asymmetry and an effective chemical shift anisotropy half of that of thionphospholipids organized in bilayers which is caused by fast lateral diffusion of the lipids around the cylinders of the hexagonal H_II phase as has been observed for the corresponding phosphatidylethanolamines. (4) Since the ³¹P-NMR resonance of the thionphospholipids is completely separated from that of natural phospholipids, these lipids can be used to study by ³¹P-NMR the motional and structural properties of individual lipids in mixed systems. This is demonstrated for various lipid mixtures in which non-bilayer lipid structures have been induced by variations in composition, temperature and presence of divalent cations. It is shown that bilayer → non-bilayer transitions can be modulated by gel → liquid crystalline phase transitions and that typical bilayer forming lipids can be incorporated into non-bilayer structures such as the hexagonal H_II phase.     

Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.     

Capturing the nanoscale complexity of cellular membranes in supported lipid bilayers

The lateral mobility of cell membranes plays an important role in cell signaling, governing the rate at which embedded proteins can interact with other biomolecules. The past two decades have seen a dramatic transformation in understanding of this environment, as the mechanisms and potential implications of nanoscale structure of these systems has become accessible to theoretical and experimental investigation. In particular, emerging micro- and nano-scale fabrication techniques have made possible the direct manipulation of model membranes at the scales relevant to these biological processes. This review focuses on recent advances in nanopatterning of supported lipid bilayers, capturing the impact of membrane nanostructure on molecular diffusion and providing a powerful platform for further investigation of the role of this spatial complexity on cell signaling.     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

Surface effects in preparation of cell-size liposomes

Effects of surface type and area were shown to be important in the yield of cell-size liposomes, but not in determining their size. The liposomes were prepared by dissolving lipids in a chloroform-methanol solution and then evaporating the solvent under nitrogen in the presence of glass beads. After evaporation of the solvent, which was rapid due to the increased surface area, the dried lipids were then swollen in water at high temperatures (higher than the phase transition of the lipids), which led to formation of giant liposomes. The number of liposomes prepared in the presence of pyrex glass beads, which increase more than 100-times the surface area of lipid-glass contact, is more than 5-times larger than in the control experiments without glass beads. The yield of liposomes in the presence of another type of glass bead was almost the same as in the control experiments. These effects may be due to long- and short-range intermolecular interactions in the glass/water/lipid system.