Planar Lipid Films Research Articles

Electrochemical Properties of Lipid Membranes Self-Assembled from Bicelles.

Supported lipid membranes are widely used platforms which serve as simplified models of cell membranes. Among numerous methods used for preparation of planar lipid films, self-assembly of bicelles appears to be promising strategy. Therefore, in this paper we have examined the mechanism of formation and the electrochemical properties of lipid films deposited onto thioglucose-modified gold electrodes from bicellar mixtures. It was found that adsorption of the bicelles occurs by replacement of interfacial water and it leads to formation of a double bilayer structure on the electrode surface. The resulting lipid assembly contains numerous defects and pinholes which affect the permeability of the membrane for ions and water. Significant improvement in morphology and electrochemical characteristics is achieved upon freeze–thaw treatment of the deposited membrane. The lipid assembly is rearranged to single bilayer configuration with locally occurring patches of the second bilayer, and the number of pinholes is substantially decreased. Electrochemical characterization of the lipid membrane after freeze–thaw treatment demonstrated that its permeability for ions and water is significantly reduced, which was manifested by the relatively high value of the membrane resistance.

Characterization of planar biomimetic lipid films composed of phosphatidylethanolamines and phosphatidylglycerols from Escherichia coli

We have characterized planar lipid films composed of phosphatidylethanolamines (PE) and phosphatidylglycerols (PG) from E. coli bacteria. The nature of the interactions and miscibility of PE and PG components within mixed lipid films was evaluated based on surface pressure measurements and Brewster angle microscopy imaging at the air-water interface. We have found that PE and PG components show tendency to form separated domains at surface pressures relevant for biological membranes. Further, we have directly compared mechanisms of formation of supported lipid bilayers either on mica or Au(111) by spreading of small unilamellar vesicles. The bilayer formation was monitored by in situ atomic force microscopy imaging. The pathways of the vesicles spreading on each substrate are substantially different and the buildup of the bilayer on Au(111) occurs through complex multistep mechanism. The morphology and nanomechanical properties of the resulting PE/PG bilayers were thoroughly compared. We have found that the interactions between lipids and supporting substrate significantly affect molecular organization within the films since the bilayer on Au(111) is uniform in terms of the topography, while the same lipid composition on mica results in formation of distinct gel and liquid disordered domains. Different molecular organization affects also nanomechanical properties of lipid films. The latter were expressed in terms of Young's moduli and bending stiffness.

Voltage Dependence of Proton Pumping by Bacteriorhodopsin Is Regulated by the Voltage-Sensitive Ratio of M 1 to M 2

The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range −160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M → bR decay of ∼5 ms ( τ 1) and ∼20 ms ( τ 2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with τ 3 ≈ 300 ms at neutral pH is observed. The fast processes ( τ 1, τ 2) can be assigned to the decay of M 2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas τ 3 is due to the decay of M 1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M 1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M 1 and the M 2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.

Interaction of Cytoskeletal Proteins with Membrane Lipids

Rapid and significant progress has been made in understanding lipid/protein interactions involving cytoskeletal components and the plasma membrane. Covalent and noncovalent lipid modifications of cytoskeletal proteins mediate their interaction with lipid bilayers. The application of biophysical techniques such as differential scanning colorimetry, neutron reflection, electron spin resonance, CD spectroscopy, nuclear magnetic resonance, and hydrophobic photolabeling, allow various folding stages of proteins during electrostatic adsorption and hydrophobic insertion into lipid bilayers to be analyzed. Reconstitution of proteins into planar lipid films and liposomes help to understand the architecture of biological interfaces. During signaling events at plasma membrane interfaces, lipids are important for the regulation of catalytic protein functions. Protein/lipid interactions occur selectively and with a high degree of specificity and thus have to be considered as physiologically relevant processes with gaining impact on cell functions.

Two-Dimensional Structure of Membrane-Bound Annexin V at 8 Å Resolution

Two-dimensional (2-D) crystals of annexin V, grown by specific binding to phosphatidylserine-containing planar lipid films, were studied by electron image analysis. Images of negatively stained two-dimensional crystals showed diffraction peaks extending to 11 Å. After correcting lattice distorsions and averaging over several crystalline areas, the resolution of the analysis was extended up to 8 Å. Observed along a direction perpendicular to the membrane plane, the four homologous domains characteristic of annexin V exhibit a noticeable difference in their distribution of protein density. An unambiguous assignment of the domains was possible due to the similar packing of annexin V molecules in the 2-D crystals and in a 3-D crystal form with pseudo- R3 symmetry. The domains I and IV (numbering according to Huber et al., Embo J. , 1990, 9, 3867-3874) appear well resolved. On the other hand, the two other domains, II and III, present an almost continuous density, with a protrusion extending outwards the annexin V molecule. In addition, no hydrophilic opening is resolved at the center of the molecule, yet a stain-filled 13-Å structure is present, surrounded by domains I, II, and IV and distant by 5 Å from the center of the molecule. We interpret these structural features as reflecting a conformational change in the annexin V structure resulting from its membrane binding.

Structure of membrane-bound human factor Va

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, γ = 107°. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm × 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.

Two-dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography*1

Two-dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography*1

Two‐dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography*

Electron crystallography constitutes a powerful new method for determining the structure of biological macromolecules. This method is best adapted to the study of ordered assemblies of macromolecules, and principally to two-dimensional (2-D) crystals of proteins. Obtaining protein 2-D crystals ordered at high resolution constitutes the major limiting step in the application of this approach. Considerable interest has been raised by the development of a rational method of 2-D crystallization based on the specific binding of proteins to planar lipid films. The applicability of this method is quasi-general in the case of soluble proteins. Its basic principles, together with examples taken from work in our group, are presented here.

A 9 Å two-dimensional projected structure of cholera toxin B-subunit-G M1 complexes determined by electron crystallography

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside G M1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 Å, while electron images diffract to 8 Å. A two-dimensional projected structure of cholera toxin B-subunit-G M1 complex has been calculated at 9 Å resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(±1) Å, b = 123(±1) Å, γ = 90 °. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 Å and 11 Å from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral β-sheet arrangement and the central α-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point-mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild-type BR, the M formation in BR Asp85---Glu is accwelerated approximately 10-fold, whereas the M decay in BR Asp96---Asn is slowed down approximately 50-fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85---Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96---Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

Optical determination of the thickness of single planar thin lipid films using the symmetrical three-layer model

The total thickness of thin lipid films can be determined by the method of absolute or relative reflectance measurements. The film is described by the symmetrical three-layer model. By assuming equal refractive indices for the hydrocarbon layer and for the organic solution, the thickness and refractive index of the head group layer can be estimated.

Voltage-dependent pore activity of the peptide alamethicin correlated with incorporation in the membrane: salt and cholesterol effects

Strong aggregation of incorporated alamethicin in the bilayer of lipid vesicles has been observed spectroscopically at aqueous peptide concentrations above a critical value c ∗ . On the other and, in conventional gating studies with planar lipid films, the onset of conducting pore formation can be characterized by a threshold voltage V ∗ . We present experimental evidence of a direct correspondence between the effects on c ∗ and V ∗ when these parameters are modulated by adding NaCl (to the aqueous medium) or cholesterol (to the lipid moiety). A quantitative analysis supports the idea that the measured aggregation actually results in pore formation, the voltage-dependence being due to an electric field effect on the partition equilibrium of the peptide between the aqueous and the lipid phases.

Alamethicin incorporation in lipid bilayers: a thermodynamic study.

Interaction of the peptide antibiotic alamethicin with phospholipid vesicles has been monitored by changes in its circular dichroic and fluorescent properties. The data are consistent with an incorporation of the peptide in the lipid bilayer. Aggregation of alamethicin in the membrane phase is evident from a characteristic concentration dependence of the incorporation, reflecting the existence of a critical concentration. The data can be fully understood in terms of a theoretical approach that includes aggregation and thermodynamic nonideality. Thermodynamic parameters of the peptide-lipid interaction have been evaluated under a variety of conditions of temperature, ionic strength, and lipid type (saturated and unsaturated fatty acid chains). From the results obtained in this study, one can extrapolate to the incorporation behavior of alamethicin at low concentrations, as they are typical for measurements of conductance across planar lipid films. This leads to a simple explanation of the voltage-gating mechanism of alamethicin in a straightforward way.

Purified lens junctional protein forms channels in planar lipid films.

Junctions isolated from bovine lenses were solubilized with the detergent octyl glucoside, and their protein(s) was reconstituted in unilamellar vesicles. The protein(s) appears as annular-shaped intramembrane particles approximately equal to 10 nm in diameter on the vesicles' fracture faces. The addition of the vesicle-containing junctional protein(s) to both sides of preformed lipid films induced voltage-dependent channels. The channels have a conductance of 200 pS in 0.1 M salt solutions and are thus large enough to account for the electrical coupling observed between intact lens fibers; they turn off when the magnitude of the voltage is increased and in the presence of octanol. Although the identity of the reconstituted channels as the communicating pathway between lens fibers remains to be proven, it is most likely that the reconstituted channels are formed by MIP-26, the major protein component of the isolated lens junctions.

Chaotropic anions and the surface potential of bilayer membranes

The chaotropic anions perchlorate and thiocyanate adsorb to artificial phospholipid membranes. The negative electrostatic potential they produce at the surface of the membranes was measured by two independent techniques. The conductance produced by neutral carriers of cations and anions was measured to estimate changes in the surface potentials of planar black lipid films and the electrophoretic mobility of phospholipid vesicles was used to monitor changes in the zeta potentials of spherical bilayer membranes. Qualitatively similar results were obtained with the two techniques. The results, moreover, agreed with the change in surface potential produced by these anions at an air water interface, as measured directly with an ionizing electrode (Randles, J.E.B. (1957) Discuss. Faraday Soc. 24, 194–199). The results obtained with artificial bilayers may explain the observation (Wieth, J.O. (1970) J. Physiol. 207, 581–609) that thiocyanate increases the sodium or potassium and decreases the sulfate permeability of erythrocyte membranes.