Chain length and pressure dependence of lipid translational diffusion

The translational diffusion of pyrene, pyrene butyric acid and pyrene decanoic acid has been determined in phosphatidylcholine bilayers of different chain length and under pressure up to 200 bars. In the liquid crystalline phase and at a given temperature the diffusion decreases with increasing chain length. At a constant reduced temperature, T _red (about 10 K above the transition temperature), long chain lipids exhibit the fastest diffusion which is in disagreement with hydrodynamic models but favours free volume models for diffusion in lipid bilayers. The volume of activation, V _act, calculated from the decrease of the diffusion coefficient with pressure, ln D/P, depends on lipid chain length. V _act decreases with decreasing lipid chain length at a given temperature, T=65°C, and increases at the reduced temperature. These results are again in agreement with the dependence of the diffusion on lipid chain length and therefore with the free volume model.Abbreviations DLPC Dilauroylphosphatidylcholine - DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmitoylphosphatidylcholine - DSPC Distearoylphosphatidylcholine - LUV Large unilamellar vesicles - SUV Small unilamellar vesicles - Tris Tris(hydroxymethyl)aminomethan     

Surface balance study of the interaction between microorganisms and lipid monolayer at the air/water interface.

Using the surface balance technique, we have compared the interaction between Acholeplasma laidlawii and some marine bacteria towards different types of monolayered lipid films. Cells from A. laidlawii and Serratia marinorubra penetrate the film, whereas cells from Psuedomonas fluorescens form a layer underneath the film. The forces that bind microorganisms to the air/water interface are not strong enough to scatter a condensed monolayer but increase the strength of loosely packed monolayers.     

Polarization-modulated FTIR spectroscopy of lipid/gramicidin monolayers at the air/water interface

Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Effect of lipid monolayers on diffusion of oxygen through the air/water interface

The dependence of the diffusion current on the depth of immersion of the electrode was studied by polarography using an open platinum electrode. As the electrode was brought from the depth of the liquid phase to its surface, an increase in the current under aerobic conditions was observed, due to diffusion of oxygen through the interface. The formation of lipid monolayers of phosphatidylcholine, stearic acid, hexadecanol, octadecanol, eicosanol, and docosanol on the water surface led to a decrease in diffusion current; the effect being most pronounced at a minimal depth of immersion of the electrode. The maximum value of the relative decrease in diffusion current R was obtained for docosanol monolaers. It was shown that the R value increases with increasing surface pressure in monolayers of phosphatidylcholine and stearic acid. It is assumed that the decrease in diffusion flow of O2 in the presence of monolayers is caused by the formation of an energy barrier that prevents the sorption of O2, which is related to the presence of hydrocarbon chains weakly interacting with oxygen.     

Effect of glycyrrhetinic acid on lipid raft model at the air/water interface

To investigate an interfacial behavior of the aglycon of glycyrrhizin (GC), glycyrrhetinic acid (GA), with a lipid raft model consisting of equimolar ternary mixtures of N-palmitoyl sphingomyelin (PSM), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL), Langmuir monolayer techniques were systematically conducted. Surface pressure (π)–molecular area (A) and surface potential (ΔV)–A isotherms showed that the adsorbed GA at the air/water interface was desorbed into the bulk upon compression of the lipid monolayer. In situ morphological analysis by Brewster angle microscopy and fluorescence microscopy revealed that the raft domains became smaller as the concentrations of GA in the subphase (C_GA) increased, suggesting that GA promotes the formation of fluid networks related to various cellular processes via lipid rafts. In addition, ex situ morphological analysis by atomic force microscopy revealed that GA interacts with lipid raft by lying down at the surface. Interestingly, the distinctive striped regions were formed at C_GA = 5.0 μM. This phenomenon was observed to be induced by the interaction of CHOL with adsorbed GA and is involved in the membrane-disrupting activity of saponin and its aglycon. A quantitative comparison of GA with GC (Sakamoto et al., 2013) revealed that GA interacts more strongly with the raft model than GC in the monolayer state. Various biological activities of GA are known to be stronger than those of GC. This fact allows us to hypothesize that differences in the interactions of GA/GC with the model monolayer correlate to their degree of exertion for numerous activities.     

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.     

Bacterial activity at the air/water interface

Microbial Ecology - By using substrate molecules of varying degrees of surface activity, we were able to measure some features of bacterial activity in the surface microlayers (SM) and in the...     

Chain length dependence of apomyoglobin folding: structural evolution from misfolded sheets to native helices

Very little is known about how protein structure evolves during the polypeptide chain elongation that accompanies cotranslational protein folding. This in vitro model study is aimed at probing how conformational space evolves for purified N-terminal polypeptides of increasing length. These peptides are derived from the sequence of an all-alpha-helical single domain protein, Sperm whale apomyoglobin (apoMb). Even at short chain lengths, ordered structure is found. The nature of this structure is strongly chain length dependent. At relatively short lengths, a predominantly non-native beta-sheet conformation is present, and self-associated amyloid-like species are generated. As chain length increases, alpha-helix progressively takes over, and it replaces the beta-strand. The observed trends correlate with the specific fraction of solvent-accessible nonpolar surface area present at different chain lengths. The C-terminal portion of the chain plays an important role by promoting a large and cooperative overall increase in helical content and by consolidating the monomeric association state of the full-length protein. Thus, a native-like energy landscape develops late during apoMb chain elongation. This effect may provide an important driving force for chain expulsion from the ribosome and promote nearly-posttranslational folding of single domain proteins in the cell. Nature has been able to overcome the above intrinsic misfolding trends by modulating the composition of the intracellular environment. An imbalance or improper functioning by the above modulating factors during translation may play a role in misfolding-driven intracellular disorders.     

Spreading of membranes at the air/water interface

This article presents an original technique for spreading membranes at the air/water interface. We have characterized enzymatically lipoprotein films d     

Spreading of liposomes at the air/water interface

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.     

Coniferyl alcohol reactivity at the air/water interface

In order to investigate the sensitivity of the lignin monomer coupling reactions to the environment physicochemical conditions, coniferyl alcohol (CA) was polymerised at the air/water interface. Characterisation of the interface during the reaction by surface pressure measurement and ellipsometry demonstrates that the reaction occurs near or at the interface. Coupling products were analysed by HPLC and compared to reaction products obtained in the case of polymerisation in solution. Relative proportions of beta-beta and beta-O-4 dehydrodimers were found to increase in air/water interface experiment.     

Spreading of biomembranes at the air/water interface.

This paper presents the compression isotherms obtained by spreading membranes of intestinal brush border, human erythrocyte and Escherichia coli (cytoplasmic) at the air/water interface. Unilamellar membrane films were formed, with a good yield, at zero surface pressure, whereas multilamellar structures were formed at high surface pressure. Once formed, the films were particularly stable and could be manipulated without any detectable loss. With doubly-labelled E. coli cytoplasmic membrane, we could show that phospholipids and proteins spread, with the same yield, as a single unit. Moreover, we studied the influence of hydrolytic enzymes, chemical agents and cations on the compression isotherm of biomembranes. The resultant changes in architecture of membrane films can provide a very simple method of studying the influence of membrane packing on catalytic activity and protein conformation of membrane-bound proteins.     

Adsorption and desorption studies of phospholipid at the air/water interface

The desorption and adsorption properties of phosphatidylserine (extracted from beef brain) at the air/water interface were studied through surface pressure measurements. The rate of dissolution of phosphatidylserine monolayer into the underlying water of natural pH is extremely slow at room temperature but increases rather suddenly around 40°C. This sudden increase of dissolution rate might be explained as the Kraft point phenomena analogous to the ionic surfactants.     

Orientation of beta-barrel proteins OmpA and FhuA in lipid membranes. Chain length dependence from infrared dichroism

The outer-membrane proteins OmpA and FhuA of Escherichia coli are monomeric beta-barrels of widely differing size. Polarized attenuated total reflection infrared spectroscopy has been used to determine the orientation of the beta-barrels in phosphatidylcholine host matrices of different lipid chain lengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples are used to deduce the strand tilt (beta = 46 degrees for OmpA and beta = 44.5 degrees for FhuA). These values are then used to deduce the order parameters, P(2)(cos alpha), of the beta-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the beta-barrels and their assembly in the membrane are discussed in terms of hydrophobic matching with the lipid chains.     

Interaction between a growth-hormone releasing hexapeptide and phospholipids spread as monolayers at the air/water interface

The interaction between a growth-hormone releasing hexapeptide and phospholipids was studied on mixed monolayers models by means of surface fluorescence. When in a monolayer this hexapeptide which contains two tryptophan molecules was observed to fluoresce. Isothermal compression experiments showed that the complex was destroyed upon compression in the case of phosphatidylethanolamine. With phosphatidylglycerol it was observed to be stable but a dramatic reversible decrease in emission was observed at high surface pressure. This is indicative of a reversible change in the organization of the peptide-phospholipid complex. These observations indicate that, in the complex, hydrophobic interactions were weak but electrostatic ones, when present, were strong enough to maintain the GHRP attached to the monolayer and not to destabilize it. The integrity of the lipid monolayer appeared not to be affected by the peptide.     

Miscibility of DPPC and DPPA in monolayers at the air/water interface

Monolayers of mixtures of 1,2-dipalmitoylphosphatidylcholine (DPPC) as the substrate and 1,2-dipalmitoylphosphatidic acid (DPPA) as the product of the hydrolysis reaction catalyzed by phospholipase D (PLD) were investigated in the presence of Ca2+. The miscibility behavior and the microstructure of mixed domains have been studied by grazing incidence X-ray diffraction (GIXD), Brewster angle microscopy and film balance measurements. The phase diagram reveals partial miscibility on both sides and a wide miscibility gap, which becomes narrower at high pressure. At low pressure, the segregation of condensed DPPA-rich domains in a fluid-like DPPC matrix was detected already at small DPPA concentrations and their structure was determined. A small amount of DPPC mixed into the segregated DPPA domains induces the transformation from rectangular to an oblique unit cell and increases the tilt angle in the condensed domains. At high pressure, two types of condensed phase domains were found: DPPC-rich and DPPA-rich. A drastic reduction of the tilt angle in the DPPC-rich domains with increasing amount of DPPA was observed. The decrease of the tilt angle must be connected with a change of the head group conformation of DPPC in such mixed domains.     

Second harmonic generation of glucose oxidase at the air/water interface

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) chromophores embedded in the GOx protein. Because of the subsequent resonance enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqueous concentrations as low as 70 nM. The protein adsorption was followed, and, at high GOx coverage, a change in the orientation of the FAD chromophore was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the biological pH of 7, its interactions with charged surfactants were also investigated. As expected, spreading positively charged surfactants onto a partial protein monolayer was found to increase the GOx surface concentration, whereas in the case of negatively charged surfactants, the GOx surface concentration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearrangement or reorientation of the protein was also observed.     

Molecular organization of the human serotonin transporter at the air/water interface

The serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10,400 and 7,100 A(2)/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7,100 A(2)/molecule was found to be approximately equal to 84 A and coincided well with the theoretical value estimated from the calculations based on the dimensions of alpha-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths d(z)=83--85 A and d(y) or d(x)=41--47 A.     

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.