Phase behavior of a model bilayer membrane with coupled leaves

We discuss the thermodynamic behavior of a bilayer composed of two coupled leaves and derive the Gibbs Phase Rule for such a system. A simple phenomenological model of such a system is considered in which the state of the bilayer is specified by the relative number of ordering lipids in the outer leaf, and in the inner leaf. Two cases are treated. In the first, both inner and outer leaves could undergo phase separation when uncoupled from one another. The bilayer can exist in four different phases, and can exhibit three-phase coexistence. In the second case, an outer layer which can undergo phase separation by itself is coupled to an inner leaf which cannot. We find that when the coupling is weak, the bilayer can exist in only two phases, one in which the outer layer is rich in ordering lipids and the inner leaf is somewhat richer in them than when uncoupled, and another in which the outer layer is poor in ordering lipids and the inner leaf is poorer in them than when uncoupled. Increasing the coupling increases the effect on the inner leaf composition due to small changes in those of the outer leaf. For sufficiently large coupling, a phase transition occurs and the bilayer exhibits four phases as in the first case considered. Our results are in accord with several observations made recently.     

Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane

Secretion of beta-lactamase was studied in Salmonella typhimurium infected with P22 phage carrying wild-type and mutant alleles of the structural gene. Cellular location of precursor and mature products of wild-type and temperature-sensitive and chain-terminating mutants was analyzed by cell fractionation and by trypsin accessibility in intact and lysed spheroplasts. The precursors of wild-type and all these mutants (none of which alter the signal peptide) are found sequestered within the cell, while all the mature forms have at least partially been translocated across the inner membrane. Thus most beta-lactamase molecules traverse the membrane after completion of their translation. It seems that the carboxyl terminus of beta-lactamase is not required for translocation across the inner membrane but is required for the protein to appear in the periplasm as a soluble species.     

Construction and optimisation of a computer model for a bacterial membrane

The main steps in the construction of a computer model for a bacterial membrane are described. The membrane has been built of 72 lipid molecules, 54 of which being 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (POPE) and 18--1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl-rac-glycerol (POPG) molecules (thus in the proportion of 3:1). The membrane was hydrated with 1955 water molecules (approximately 27 water molecules per lipid). To neutralise the electronic charge (-e) on each POPG molecule, 18 sodium ions (Na+) were added to the membrane close to the POPG phosphate groups. The atomic charges on the POPE and POPG headgroups were obtained from ab initio quantum mechanical restrained electrostatic potential fitting (RESP) (Bayly et al., 1993, J. Phys. Chem. 97, 10269) using the GAMESS program at the 6-31G* level (Schmidt et al., 1993, J. Comput. Chem. 14, 1347). The model constructed in this way provided an initial structure for subsequent molecular dynamics simulation studies intended to elucidate the atomic level interactions responsible for the structure and dynamics of the bacterial membrane.     

The ferret as a model for inner ear research

Viral infections have long been suspected to be causative agents in a number of inner ear dysfunctions. With few exceptions, the virus has not been demonstrated as the direct agent leading to hearing loss and/or vertigo. Selective inner ear changes have been observed recently in sensory and nonsensory epithelial cells in the ferret model for Reye's syndrome after intranasal inoculation with influenza B combined with aspirin administration and the creation of an arginine deficiency. Such findings suggest that these agents act synergistically on the inner ear, particularly on cells that are metabolically active, and that the ferret may now be a useful model to examine the role of certain upper respiratory tract viruses implicated in inner ear disorders, singly and in combination with other agents that may cause metabolic alterations.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.     

Building a bilayer model of the neuromuscular synapse

Progress over the past 10 years has made it possible to construct a simple model of neurotransmitter release. Currently, some models use artificially formed vesicles to represent synaptic vesicles and a planar lipid bilayer as a presynaptic membrane. Fusion of vesicles with the bilayer is via channel proteins in the vesicle membrane and an osmotic gradient. In this paper, a framework is presented for the successful construction of a more complete model of synaptic transmission. This model includes real synaptic vesicles that fuse with a planar bilayer. The bilayer contains acetylcholine receptor (AChR) channels which function as autoreceptors in the membrane. Vesicle fusion is initiated following a Ca²⁺ flux through voltage-gated Ca²⁺ channels. Key steps in the plan are validated by mathematical modeling. Specifically, the probability that a reconstituted AChR channel opens following the release of ACh from a fusing vesicle, is calculated as a function of time, quantal content, and number of reconstituted AChRs. Experimentally obtainable parameters for construction of a working synapse are given. The inevitable construction of a full working model will mean that the minimal structures necessary for synaptic transmission are identified. This will open the door in determining regulatory and modulatory factors of transmitter release.     

Adriamycin as a probe for the transversal distribution of cardiolipin in the inner mitochondrial membrane

The ability of adriamycin to complex cardiolipin was used to determine the distribution of cardiolipin across the inner membrane of rat liver and heart mitochondria. In both mitochondrial types, about 57 +/- 5% of the total cardiolipin was found to be located in the cytoplasmic face of the inner membrane. Mitochondria and mitoplasts were used to study the cytoplasmic face of the inner membrane, purified submitochondrial vesicles with inverted membrane orientation for the matrix face. The cardiolipin amount titrated by adriamycin in the latter was found to be complementary to the amount titrated in the cytoplasmic face. The adriamycin association constant determined for the first saturation level of mitochondria was in good agreement with the value published by Goormaghtigh et al. (Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M. (1980) Biochim. Biophys. Acta 597, 1-14) for cardiolipin in artificial membranes. Two binding plateaus were observed when increasing amounts of adriamycin were added to mitochondria. The plateau at higher concentrations is conveniently explained by the penetration of adriamycin into mitochondria and the titration of cardiolipin in the matrix face. Scatchard plot analysis of the binding curves leading to the two plateaus produced almost identical association constants. The total amount of cardiolipin in mitochondria calculated from curves of this type corresponded to the total amount of cardiolipin determined by phosphate analysis of extracts, analyzed by thin layer chromatography.     

The elastic deformability of closed multilayered membranes is the same as that of a bilayer membrane

The elastic behavior of closed multilayered membranes is analyzed with the assumption that the constituent layers are in close contact but are unconnected in the sense that they are free to slide by one another. The system exhibits three independent elastic deformation modes for any number of the constituent layers equal to or larger than two. These are the area expansivity of the membrane neutral surface, and the local and non-local membrane bending. The corresponding elastic moduli are expressed in terms of the elastic moduli of the constituent layers, their areas, and distances between their neutral surfaces. Closed multilayered membranes only differ from a closed bilayer membrane in that for any of their shapes some of the constituent layers are expanded and some compressed.     

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1–10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35°C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421–422). The predicted water permeability coefficient agrees with the measured value at 32°C but is 40% above the measured value at 20°C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 ± 0.3 kcal/mol, while the measured value is 14.2 ± 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Monolayers at the oil/water interface as a proper model for bilayer membranes

In order to clarify the structural relationship between lipid monolayer and bilayer membranes, physical states of these membranes are discussed from their energetic points of view. It is concluded that the monolayer formed at the oil/water interface is a proper model system to represent the physical state of half of a bilayer in its liquid crystalline state. The theoretical prediction is confirmed by the monolayer surface tension measurements and the bilayer conductance experiments with water soluble (extrinsic) proteins. It is also deduced that the surface pressure of the bilayer in the liquid crystalline state is quite high, about 45 dyn/cm, and the interaction of cytochrome c with the bilayer is mainly electrostatic at the bilayer membrane periphery.     

Interlayer-interaction for a lipid bilayer model

In order to elucidate the molecular basis of stability of lipid bilayer membranes, a system of two parallel monolayers consisting of oriented hydrocarbons with polar groups, which are oriented outwardly, is investigated. Relying upon the second-order perturbation theory of intermolecular forces between asymmetric molecules, a general expression for interlayer interaction energies is obtained for induced dipole-induced dipole, dipoleinduced dipole and dipole-dipole interactions. Using the polarizability tensor for a hydrocarbon unit C₂H₄, calculated after Smith & Mortensen (1960), and assuming 3 Debye unit for a dipole moment of a polar group, numerical estimations are performed.     

Visualization of domain formation in the inner and outer leaflets of a phospholipid bilayer

Large vesicles (5-10-micron in diameter) were formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera and digital image processor. When such vesicles contained a fluorescent phosphatidic acid (PA) and were exposed to 2 mM CaCl2 or 0.5 mM PrCl3, it was possible to visualize PA-enriched domains within the vesicles. Calcium-induced domain formation was reversible in the presence of 4 mM EGTA. Vesicles were formed containing fluorescent PA on either the inner or outer leaflet of the bilayer and the patching and dissolution of patching were studied under conditions where calcium was present on the outside of the vesicle and where calcium was distributed across the bilayer. In addition, vesicles were formed with two different fluorescent PA's, one on the inner leaflet and a different one on the outer leaflet of the bilayer. The results of the experiments show that in vesicles formed primarily with naturally occurring phospholipids such as egg phosphatidylcholine or brain phosphatidylethanolamine, there was no coordinate action of the two leaflets of the bilayer. An exception to this was found, however, if the vesicles were formed in the presence of primarily dioleoyl phospholipids (greater than 95 mol %). In these vesicles there was a coordinate or coupled response to calcium by the two leaflets of the bilayer. In most cases, however, the two leaflets of the bilayer showed independent or uncoupled domain formation.     

Alignment of cholesterol in the membrane bilayer

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by lines. The FCC and lines are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in rhombs, lines only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and lines, as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.     

Rapid renaturation of denatured and aggregated proteins using liquid paraffin as a pseudolipid bilayer membrane

A means of rapidly renaturing denatured protein was devised and evaluated. Three liquids were laminarly layered in a centrifuge tube, in which two solutions sandwiched liquid paraffin so as to form a pseudolipid bilayer. Denatured and aggregted protein placed on the upper surface of liquid paraffin was renatured as it passed through liquid-paraffin layer into the renaturation buffer during the centrifugation. The aggregated and denatured protein selectively passed through the liquid-paraffin layer, whereas other solutions, such as chaotropic agents or organic solvent, could not. This means that a rapid dilution condition favorable for protein renaturation was realized in a small scale. Aggregated and denatured BSA and ribonuclease A were renatured and resolubilized as they passed through the liquid-paraffin layer into an appropriate renaturation buffer solution. This method was also applied to the rapid heme reconstitution of myoglobin from Feprotoporphyrin IX to Zn-protoporphyrin IX. (c) 1994 John Wiley & Sons, Inc.