Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation

Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.     

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Topology and acylation of spiralin.

Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, approximately 15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain.     

Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.     

The molecular mechanism of monolayer-bilayer transformations of lung surfactant from molecular dynamics simulations

The aqueous lining of the lung surface exposed to the air is covered by lung surfactant, a film consisting of lipid and protein components. The main function of lung surfactant is to reduce the surface tension of the air-water interface to the low values necessary for breathing. This function requires the exchange of material between the lipid monolayer at the interface and lipid reservoirs under dynamic compression and expansion of the interface during the breathing cycle. We simulated the reversible exchange of material between the monolayer and lipid reservoirs under compression and expansion of the interface. We used a mixture of dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol, cholesterol, and surfactant-associated protein C as a functional analog of mammalian lung surfactant. In our simulations, the monolayer collapses into the water subphase on compression and forms bilayer folds. On monolayer reexpansion, the material is transferred from the folds back to the interface. The simulations indicate that the connectivity of the bilayer aggregates to the monolayer is necessary for the reversibility of the monolayer-bilayer transformation. The simulations also show that bilayer aggregates are unstable in the air subphase and stable in the water subphase.     

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

Structure and orientation of lung surfactant SP-C and L-alpha-dipalmitoylphosphatidylcholine in aqueous monolayers.

SP-C, a pulmonary surfactant-specific protein, aids the spreading of the main surfactant phospholipid L-alpha-dipalmitoylphosphatidylcholine (DPPC) across air/water interfaces, a process that has possible implications for in vivo function. To understand the molecular mechanism of this process, we have used external infrared reflection-absorption spectroscopy (IRRAS) to determine DPPC acyl chain conformation and orientation as well as SP-C secondary structure and helix tilt angle in mixed DPPC/SP-C monolayers in situ at the air/water interface. The SP-C helix tilt angle changed from approximately 24 degrees to the interface normal in lipid bilayers to approximately 70 degrees in the mixed monolayer films, whereas the acyl chain tilt angle of DPPC decreased from approximately 26 degrees in pure lipid monolayers (comparable to bilayers) to approximately 10 degrees in the mixed monolayer films. The protein acts as a "hydrophobic lever" by maximizing its interactions with the lipid acyl chains while simultaneously permitting the lipids to remain conformationally ordered. In addition to providing a reasonable molecular mechanism for protein-aided spreading of ordered lipids, these measurements constitute the first quantitative determination of SP-C orientation in Langmuir films, a paradigm widely used to simulate processes at the air/alveolar interface.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

Infrared reflection-absorption of melittin interaction with phospholipid monolayers at the air/water interface.

The interaction of melittin with monolayers of 1,2-dipalmitoylphosphatidylcholine and 1,2-dipalmitoylphosphatidylserine has been investigated with infrared external reflection-absorption spectroscopy. Improved instrumentation permits determination of acyl chain conformation and peptide secondary structure in situ at the air/water interface. The IR frequency of the 1,2-dipalmitoylphosphatidylcholine antisymmetric acyl chain CH2 stretching vibration decreases by 1.3 cm-1 upon melittin insertion, consistent with acyl chain ordering, whereas the same vibrational mode increases by 0.5 cm-1 upon peptide interaction with the 1,2-dipalmitoylphosphatidylserine monolayer, indicative of chain disordering. Thus the peptide interacts quite differently with zwitterionic compared with negatively charged monolayer surfaces. Melittin in the monolayer adopted a secondary structure with an amide l(l') frequency (1635 cm-1) dramatically different from the alpha-helical motif (amide l frequency 1656 cm-1 in a dry or H2O hydrated environment, amide l' frequency 1645 cm-1 in an H-->D exchanged alpha-helix) assumed in bilayer or multibilayer environments. This work represents the first direct in situ spectroscopic indication that peptide secondary structure in lipid monolayers may differ from that in bilayers.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

Lateral tensions and pressures in membranes and lipid monolayers

The effects of lateral tension on the properties of membranes are often explained in comparison with analogous experiments on monolayers, which yield more detailed data. To calculate the effects of changes in tension on the composition of, or incorporation of amphiphiles into membranes we examine (i) the fidelity of the monolayer analogy, (ii) the range of possible tensions in a membrane, and the way in which tensions arise and (iii) the equilibrium partitioning of amphiphiles between aqueous solution and a bilayer under tension. We argue that, at the same areas per molecule, a monolayer at an $\text{n-}\text{alkane}\text{/}\text{water}$ interface is a closer analogy of the lipid bilayer than a monolayer at an air/water interface. Next, we show from a thermodynamic argument that changes in membrane tension can affect the absorption of very large amphiphiles such as proteins, but that physiological tensions are unlikely to affect the absorption of lipids or drugs. Finally we consider the possibility that the measured bulk tension in a complicated membrane such as that of the erythrocyte may be larger than the local tension in the fluid mosaic portions, and suggest a model which explains the ability of the erythrocyte membrane to withstand much higher tensions than other biological membranes and lipid bilayers.     

Structure and dynamics of model pore insertion into a membrane

A cylindrical transmembrane molecule is constructed by linking hydrophobic sites selected from a coarse grain model. The resulting hollow tube assembly serves as a representation of a transmembrane channel, pore, or a carbon nanotube. The interactions of a coarse grain di-myristoyl-phosphatidyl-choline hydrated bilayer with both a purely hydrophobic tube and a tube with hydrophilic caps are studied. The hydrophobic tube rotates in the membrane and becomes blocked by lipid tails after a few tens of nanoseconds. The hydrophilic sites of the capped tube stabilize it by anchoring the tube in the lipid headgroup/water interfacial region of each membrane leaflet. The capped tube remains free of lipid tails. The capped tube spontaneously conducts coarse grain water sites; the free-energy profile of this process is calculated using three different methods and is compared to the barrier for water permeation through the lipid bilayer. Spontaneous tube insertion into an undisturbed lipid bilayer is also studied, which we reported briefly in a previous publication. The hydrophobic tube submerges into the membrane core in a carpetlike manner. The capped tube laterally fuses with the closest leaflet, and then, after plunging into the membrane interior, rotates to assume a transbilayer orientation. Two lipids become trapped at the end of the tube as it penetrates the membrane. The hydrophilic headgroups of these lipids associate with the lower tube cap and assist the tube in crossing the interior of the membrane. When the rotation is complete these lipids detach from the tube caps and fuse with the lower leaflet lipids.     

Structure and orientation study of fusion peptide FP23 of gp41 from HIV-1 alone or inserted into various lipid membrane models (mono-, bi- and multibi-layers) by FT-IR spectroscopies and Brewster angle microscopy

In the present work, we study the structure and the orientation of the 23 N-terminal peptide of the HIV-1 gp 41 protein (AVGIGALFLGFLGAAGSTMGARS) called FP23. The behaviour of FP23 was investigated alone at the air/water interface and inserted into various lipid model systems: in monolayer or multibilayers of a DOPC/cholesterol/DOPE/DOPG (6/5/3/2) and in a DMPC bilayer. PMIRRAS and polarized ATR spectroscopy coupled with Brewster angle microscopy and spectral simulations were used to precisely determine the structure and the orientation of the peptide in its environment as well as the lipid perturbations induced by the FP23 insertion. The infra-red results show the structural polymorphism of the FP23 and its ability to transit quasi irreversibly from an alpha-helix to antiparallel beta-sheets. At the air/water interface, the transition is induced by compression of the peptide alone and is modulated by compression and lipid to peptide ratio (Ri) when FP23 is inserted into a lipid monolayer. In multibilayers and in a single bilayer, there is coexistence in quasi equal proportions of alpha-helix and antiparallel beta-sheets of FP23 at low peptide content (Ri=100, 200) while antiparallel beta-sheets are predominant at high FP23 concentration (Ri=50). In (multi)bilayer systems, evaluation of dichroic ratios and sprectral simulations show that both the alpha-helix and the antiparallel beta-sheets are tilted at diluted FP23 concentrations (tilt angle of alpha-helix with respect to the normal of the interface=36.5+/-3.0 degrees for FP23 in multibilayers of DOPC/Chol/DOPE/DOPG at Ri=200 and 39.0+/-5.0 degrees in a single bilayer of DMPC at Ri=100 and tilt angle of the beta-sheets=36.0+/-2.0 degrees for the beta-sheets in multibilayers and 30.0+/-2.0 degrees in the lipid bilayer). In parallel, the FP23 induces an increase of the lipid chain disorder which shows both by an increase of the methylene stretching frequencies and an increase of the average C-C-C angle of the acyl chains. At high FP23 content (Ri=50), the antiparallel beta-sheets induce a complete disorganization of the lipid chains in (multi)bilayers.     

Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers

The lipid-layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D-crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP-binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed.     

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lβ (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160-2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in β-sheet contents and an increase in β-strand structures, probably related to protein-protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. II. Suppression of tetraphenylborate conductance and changes of interfacial potentials.

It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by suppression of TPhB- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decreases being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25 - 30% of the total 2,4-D-induced change of the potential difference is between the plane of absorption of TPhB- and the aqueous solution, and the remaining fraction is between the membrane interior and the absorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studied of transport of positive and negative ions across bilayer membranes. In contract, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membranes, but rather with a layer of 2,4-D molecules absorbed at the nonpolar/polar membrane boundary.     

Gap junction hemichannel interactions with zwitterionic lipid, anionic lipid, and cholesterol: molecular simulation studies

Interactions with membrane lipids can exert dramatic functional consequences on gap junction proteins. Recent experimental work has highlighted the importance of anionic lipids and cholesterol in facilitating channel activity. In this work, we have employed a coarse-grained molecular model in conjunction with molecular dynamics (MD) simulations to study the interactions between a connexin 26 (Cx26) hemichannel and a number of lipid species, including palmitoyloleoylphosphatidylcholine (POPC), anionic palmitoyloleoylphosphatidic acid (POPA), and cholesterol, in order to identify sites at the protein interface which may exhibit preferential, specific binding to these lipids, as well as determine the characteristics of these interactions. We have also employed an atomistic model of Cx26 embedded in a mixed PA/PC bilayer as a comparison and to elucidate further lipid-protein interactions. Our simulation results suggest enrichment of interfacial PA at the intracellular leaflet at high bulk PA concentrations. PC can form tight binding interactions with the hemichannel, particularly at intersubunit crevices (classical nonannular sites). In mixed bilayers, however, POPA competes with POPC for these sites, displacing the latter in some cases. While the residues responsible for interactions with PC and PA are similar, the latter exhibits a unique property of being capable of forming stable hydrophilic contacts with multiple residues spanning two different adjacent subunits at both leaflets of the bilayer, as opposed to POPC which can only do so at the extracellular side. These results suggest that POPA may be essential to channel function by acting as an intersubunit lipid bridge. Additionally, we propose that the compositional enrichment of POPA at the Cx26 interface may serve important roles in voltage gating. Simulation of a mixed POPC:cholesterol bilayer suggests that the hemichannel enhances the transbilayer mobility of vicinal cholesterols, increasing the likelihood of site-hopping and interleaflet flip-flop transitions.