Erythrosin and pH gradient induced photo-voltages in bilayer membranes

Summary Erythrosin and light flashes induce voltage transients across bilayer membranes in the presence of transmembrane pH gradients. Fast voltage transients, which rise in <50 nsec and fall in 500 nsec, are attributed to photo-deprotonation of dye sorbed in the glycerol region of phospholipid membranes. Six other halogenated xanthene dyes induce similar effects, which apparently resulted from triplet states of monoanionic dye. No photo-effects were observed with fluorescein.     

Conductivity and structural transitions in bilayer lipid membranes

5 structural transitions were found in bilayer lipid membranes (BLM) from egg lecithin (EL) within the temperature range 14-44 degrees C. In the transition zone BLM conductivity abruptly increases, in some cases current fluctuations of the order 150 pC of the channel type are initiated. The transition temperatures observed in BLM from EL coincide with those in biological membranes. The cause of this phenomenon is discussed, as well as possible use of these BLM in the region of structural transition as a model of cellular receptor to electromagnetic fields.     

Voltage transients from photo-isomerizing azo dye in bilayer membranes.

Voltage transients are induced by brief light flashed on bilayer membranes with absorbed 3,3'-bis(alpha-(trimethylammonium)methyl)azobenzene (Bis-Q). The voltages are positive for trans-to-cis photo-isomerization, and negative for cis-to-trans photo-isomerization. The risetimes in phosphatidylethanolamine-decane bilayer membranes indicate that absorbed trans-Bis-Q is photo-isomerized to cis within 2 microseconds, and that cis is photo-isomerized to trans within 15 microseconds.     

Diacylglycerol causes major structural transitions in phospholipid bilayer membranes

We demonstrate for the first time that major structural changes are imposed on various phospholipid bilayers by diacylglycerol, a product of phosphatidylinositol metabolism. By 5 mole percent in phosphatidylethanolamine a lamellar to hexagonal transition starts that is complete at 10 mole percent. At 30 mole percent it causes the same transition in phosphatidylcholine and forms a cubic phase at 80 mole percent. Diacylglycerol disorders the phosphatidylserine lamellar phase. We view the formation of the non-lamellar phases as diagnostic of the destabilizations that diacylglycerol can cause in membranes. We suggest how DAG may act both in its specific activation of membrane enzymes and in inducing membrane fusion.     

Thermal and structural behavior of natural cerebroside 3-sulfate in bilayer membranes

Differential scanning calorimetry (DSC), polarizing microscopy and X-ray diffraction studies have been performed on dry and hydrated natural bovine brain sulfatides. Dry sulfatide fractions exhibit a high temperature transition (delta H = 6.6 kcal/mol sulfatide) at 87.3 degrees C. X-ray diffraction shows this transition to be associated with a hydrocarbon chain order-disorder transformation between two lamellar phases. Hydrated sulfatide dispersions undergo a complex chain order-disorder transition (delta H = 7.5 kcal/mol sulfatide) at 32 degrees C with two peak temperatures at 35 degrees C and 47 degrees C. Structural studies performed on hydrated liquid-crystal sulfatide dispersions at 75 degrees C verify the existence of a bilayer structure over the 16 wt.% to 50 wt.% phosphate buffer (pH = 7.4) range. The interbilayer separation between galactosyl-3-sulfate groups averages 48 A as the multilamellar bilayers swell with the addition of phosphate buffer. The formation of micellar phases is not observed at high water contents. The comparison of the structural characteristics of dry and hydrated sulfatides with structural data for dry and hydrated bovine brain non-sulfated glycolipid (cerebroside) is discussed in molecular terms.     

The intrinsic structural asymmetry of highly curved phospholipid bilayer membranes

Phosphorus-31 NMR studies of solutions of small L-alpha-dipalmitoyl phosphatidylcholine bilayer vesicles containing sodium dimethyl phosphate uniformly distributed between the continuous external and the intravesicular aqueous spaces, with the paramagnetic shift reagent Pr3+ present only in the external space, are reported. These studies give the distribution both of dipalmitoyl phosphatidylcholine in the vesicle inner and outer monolayers and of dimethyl phosphate in the aqueous spaces. With the third necessary parameter obtained from the vesicle sedimentation coefficient, the very different packing parameters of dipalmitoyl phosphatidylcholine in inner and outer monolayers can be determined. The vesicle outer radius is 109 A. Although the total bilayer thickness is virtually identical to that of planar bilayers, the outer monolayer is thicker (20 A) and the inner monolayer thinner (15 A). The area per head group at the inner surface, 68 A2, is like the planar value, but the tails are much more folded, so as to decrease the radial lengths and increase the tangential spreat (to 94A2). The reverse is true in the outer layer: the surface per head group is 76 A2, tapering to 51 A2 in the tail region, so that outer layer tails are relatively extended. The difference is equivalent to a shift of about two 2g1 kinks from outer to inner layers; the uneven packing certainly affects fluidity, and may have important biological consequences.     

Thermodynamic and structural properties of phosphatidylserine bilayer membranes in the presence of lithium ions and protons

The binding of lithium ions to phosphatidylserine has been studied by differential scanning calorimetry for dialkyl and diacyl lipid forms and by X-ray diffraction for dihexadecylphosphatidylserine (DHPS). On first mixing DHPS with LiCl solutions an ordered L_β (L_c) phase is formed with a bilayer repeat distance of 5.55 nm and one strong wide-angle, chain-chain reflection at 0.405 nm (26°C), corresponding to bilayers of little, (mono)hydrated lipid with chains approximately perpendicular to the membrane surface. On heating, this phase transforms to an inverted hexagonal phase (H₁₁, H_α) with a repeat distance of 3.75 nm, at a chain-melting transition temperature of approximately 90°C (DHPS). Cooling, after equilibration of the DHPS⁻·Li⁺ sample in the fluid phase, creates a new low-temperature phase (L_c') which has a repeat distance of 4.0 nm, corresponding to strongly tilted chains (ϕ=42°). The L_c phase also transforms on heating to the H_α phase, but at a considerably lower chain-melting temperature of approx. 70°C (DHPS). The calorimetric behavior as a function of Li⁺ concentration is qualitatively very similar for the different dialkyl- and diacylphosphatidylserines studied, and is analogous to the results obtained on pH titration. After an initial small increase in transition temperature, that is caused by coulombic ion binding and concomitant surface charge neutralization, a much larger increase in the chain-melting transition temperature occurs, caused by dehydration of the lipid, as a consequence of a further stereospecific ion binding. This suggests that Li⁺ and H⁺ have similar binding sites on the PS headgroup.     

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.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Lipid-polypeptide interactions in bilayer lipid membranes

Summary The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10^–8 m polylysine (Ps⁺) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10^–8 to 10^–5 mho/cm²) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps⁺ is present, by addition of Ps⁺ to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps⁺ folds into an -helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps⁺ or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Interactions between digitonin and bilayer membranes

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.     

Conformational partitioning of the fusion peptide of HIV-1 gp41 and its structural analogs in bilayer membranes

Experiments have shown that the ability of the HIV-1 virus to infect cells can be greatly diminished by deactivation of the N-terminal (fusion) peptide of its glycoprotein gp41. Deactivation can be achieved by the deletion of several amino acid residues, or replacement of a hydrophobic residue with a polar residue, to form mutant variants of the wild-type peptide. We report Monte Carlo simulation studies of a simplified peptide/membrane model, representing the interaction of an HIV-1 fusion peptide (FP) and four closely related mutagens with a lipid bilayer. In agreement with experimental results, we show that FP inserts deeply into the bilayer at approximately 40 degrees to the bilayer normal. We also show a previously unreported behavior of membrane peptides, namely their equilibrium partitioning between several distinct conformations within the bilayer. We quantify this partitioning behavior and characterize each conformation in terms of its geometry, energy, and entropy. The diminished ability of FP mutagens to hemolyse and aggregate red blood cells due to their partitioning into unfavorable conformations, is also discussed. Our analysis supports a negative curvature mechanism for red blood cell hemolysis by FP. We also suggest that the small repulsive forces between surface-adsorbed peptides in opposing membrane surfaces may block aggregation.     

Thermodynamics of glycophorin in phospholipid bilayer membranes

We have developed a model of glycophorin in a phospholipid bilayer membrane in order to study the thermodynamics of this system and to understand the detailed behavior of recent calorimetric data. We assume that the larger glycophorin polar group can be considered as either adopting a pancakelike conformation at the bilayer interface (D state) or be directed generally away from the interface (U state) [Ruppel, D., Kapitza, H.G., Galla, H.J., Sixl, F., & Sackmann, E. (1982) Biochim. Biophys. Acta 692, 1-17]. Lipid hydrocarbon chains are described either as excited (e state) with high energy and relatively many gauche conformers or as generally extended (g state) with low energy. We performed a Monte-Carlo simulation using the Glauber and Kawasaki procedures on a triangular lattice which represents the plane of half of the bilayer. Lattice sites can be occupied either by lipid hydrocarbon chains or by model glycophorin alpha-helical hydrophobic cores. The states D and U are represented by hexagons of different sizes in the plane of the lattice, and the hard core repulsion between two such polar groups is accounted for by forbidding hexagon-hexagon overlap. We have studied the effect of having the glycophorin polar group interact in various ways with the lipid bilayer. We find that the protein polar group in its D state interacts, either directly or indirectly, with the lipid bilayer so as to reduce the effective lateral pressure acting on the lipid hydrocarbon chains by about 3 dyn/cm. Polar groups in their U states do not reduce this lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.     

Properties of lecithin-dodecaprenol macrovesicular bilayer membranes

The ionic transport properties, capacitance and breakdown voltage of bilayer macrovesicles made from lecithin, dodecaprenol and their mixtures have been studied. The electrical measurements showed that polyprenol in lipid bilayers increases membrane permeability and elasticity, and decreases membrane thickness. Some physiological implications of these findings are indicated.