Lipid Bilayer Model Membranes Research Articles

Isothermal Titration Calorimetry Studies of the Binding of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.

Isothermal Titration Calorimetry Studies of the Binding of a Rationally Designed Analogue of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and 31P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11–15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23–25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid–crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen–bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.

A molecular dynamics transition path sampling study of model lipid bilayer membranes inaqueous environments

The transversal motion of lipids across biological membranes or flip–flop in aqueous mediais of fundamental importance in understanding chemical reactions produced in cells. It isalso an example of a rare event in a biological system. Such a process has been investigatedby molecular dynamics transition path sampling, which is a powerful tool designed to dealwith rare events in a wide range of systems. The study covers a wide range of temperatureconditions and includes the analysis of flip–flop free energies together with adetailed study of vibrational densities of states associated with flip–flop motions.

Effects of Natural and Enantiomeric Cholesterol on the Thermotropic Phase Behavior and Structure of Egg Sphingomyelin Bilayer Membranes

Phospholipids, sphingolipids, and sterols are the major lipid components of the plasma membranes of eukaryotic cells. Because these three lipid classes occur naturally as enantiomerically pure compounds, enantiospecific lipid-lipid and lipid-sterol interactions could in principle occur in the lipid bilayers of eukaryotic plasma membranes. Although previous biophysical studies of phospholipid and phospholipid-sterol model membrane systems have consistently failed to observe such enantiomerically selective interactions, a recent monolayer study of the interactions of natural and enantiomeric cholesterol with egg sphingomyelin has apparently revealed the existence of enantiospecific sterol-sphingolipid interactions. To determine whether enantiospecific sterol-sphingolipid interactions also occur in more biologically relevant lipid-bilayer systems, differential scanning calorimetric, x-ray diffraction, and neutral buoyant-density measurements were utilized to study the effects of natural and enantiomeric cholesterol on the thermotropic phase behavior and structure of egg sphingomyelin bilayers. The calorimetry experiments show that the natural and enantiomeric cholesterol have essentially identical effects on the temperature, enthalpy, and cooperativity of the gel/liquid-crystalline phase transition of egg sphingomyelin bilayers within the limits of experimental error. As well, the x-ray diffraction and neutral buoyancy experiments indicate that bilayers formed from mixtures of natural or enantiomeric cholesterol and egg sphingomyelin have, within experimental uncertainty, the same structure and mass density. We thus conclude that significant enantioselective cholesterol-sphingolipid interactions do not occur in this lipid-bilayer model membrane system.

Photoinduced flip-flop of amphiphilic molecules in lipid bilayer membranes.

We demonstrate localized photoinduced flip-flop of stilbazolium markers in model lipid bilayer membranes. The flip-flop mechanism and dynamics are determined by combined two-photon excited fluorescence and second-harmonic generation microscopy. Upon illumination of labeled membranes with a femtosecond laser beam, two-photon absorption induced photoisomerization provokes a significant increase in the cis- marker population whose flip-flop rate was determined to be at least a thousand times faster than that for transmarkers, allowing the possibility of fast targeted control of the local interleaflet distribution of markers in biological membranes.

Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes.

Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively strong interactions with anionic phospholipids, may explain why GS14dK(4) retains relatively high antimicrobial activity. However, its low hemolytic activity is probably largely attributable to its low propensity to penetrate and/or partition into cholesterol-containing zwitterionic lipid membranes.

Syringotoxin pore formation and inactivation in human red blood cell and model bilayer lipid membranes

The effect of syringotoxin (ST), a member of the cyclic lipodepsipeptides family (CLPs) produced by Pseudomonas syringae pv. syringae on the membrane permeability of human red blood cells (RBCs) and model bilayer lipid membranes (BLMs) was studied and compared to that of two recently investigated CLPs, syringomycin E (SRE) and syringopeptin 22A (SP22A) [Biochim. Biophys. Acta 1466 (2000) 79 and Bioelectrochemistry 52 (2000) 161]. The permeability-increasing effect of ST on RBCs was the least among the three CLPs. A time-dependent ST pore inactivation was observed on RBCs at 20 and 37 °C but not at 8 °C. From the kinetic model worked out parameters as permeability coefficient of RBC membrane for 86Rb + and pores mean lifetime were calculated. A shorter pores mean lifetime was calculated at 37 °C then at 20 °C, which gave us an explanation for the unusual slower rate of tracer efflux measured at 37 °C then that at 20 °C. The results obtained on BLM showed that the pore inactivation was due to a decrease in the number of pores but not to a change of their dwell time or conductance.

Differential scanning calorimetry and 2H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane α-helical peptides on the organization of phosphatidylcholine bilayers

We have studied the effects of the incorporation of the α-helical transmembrane peptides Ac-K2-L24-K2-amide (L24) and Ac-K2-(L-A)12-K2-amide ((LA)12) on the thermotropic phase behavior of 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) lipid bilayer model membranes by differential scanning calorimetry (DSC) and the conformational and orientational order of the phospholipid chains by Fourier transform infrared (FTIR) spectroscopy and 2H nuclear magnetic resonance (2H-NMR) spectroscopy, respectively. Our DSC and FTIR spectroscopic studies indicate that the peptides L24 and (LA)12 both decrease the temperature and enthalpy of the gel/liquid-crystalline phase transition of DPPC-d62 bilayers, with (LA)12 having the greater effect in this regard. An examination of the frequencies of the CH2 and CD2 symmetric stretching bands of the infrared spectra of liquid-crystalline states of the peptide-free and peptide-containing DPPC-d62 and POPC-d31 samples, and a comparison with the orientational order as measured by 2H-NMR spectroscopy as well as with the chain order as measured by electron spin resonance spectroscopy, lead us to conclude that the CH2 (or CD2) stretching frequencies of lipid hydrocarbon chains are not a reliable measure of chain conformational order in lipid bilayers containing significant amounts of peptides or other lipophilic inclusions. In contrast, the results of our 2H-NMR spectroscopic studies present a consistent picture in which both L24 and (LA)12 increased in a similar way the time-averaged orientational order of the lipid chains of their liquid-crystalline lipid bilayer hosts. The comparison of the effects L24 and (LA)12 on phosphatidylcholine bilayers indicates that the gel-to-liquid-crystalline phase transition appears to be more sensitive to small changes in transmembrane peptide surface topology than hydrocarbon carbon chain orientational order in the liquid-crystalline state.

Determination of Membrane Immersion Depth with O 2: A High-Pressure 19F NMR Study

Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (P O2) of 100 Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depth-dependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF 2) 5C 2H 4-O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates ( R 1 P ) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by 19F NMR. The paramagnetic relaxation rates, R 1 P , of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R 1 P to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed.

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP)

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4°C upon heating and two exothermic transitions between 20 and 30°C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid–crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid–crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4°C, this structure converts to a liquid–crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid–crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.

Calorimetric and Spectroscopic Studies of the Thermotropic Phase Behavior of Lipid Bilayer Model Membranes Composed of a Homologous Series of Linear Saturated Phosphatidylserines

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, n-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and 31P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L β )-to-lamellar liquid crystalline (L α ) phase transition. However, after low temperature incubation, the L β /L α phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L β phase to a highly ordered lamellar crystalline (L c) phase. Upon heating, the L c phase of dilauroyl phosphatidylserine converts directly to the L α phase at a temperature slightly higher than that of its original L β /L α phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L β /L α phase transition alluded to above. However, with the longer chain compounds, the L c phase first converts to the L β phase at temperatures some 10–25°C below that at which the L β phase converts to the L α phase. Our results also suggest that shorter chain homologues form L c phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L c phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains.

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.

Using O2 to probe membrane immersion depth by 19F NMR.

A fluorinated detergent, CF(3)(CF(2))(5)C(2)H(4)-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by (19)F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on (19)F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (R(1)) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O(2) at least 100-fold (i.e., P(O(2)) greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (R(1)(20bar) - R(1)(0)) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(N,N-dimethyl-N-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in (19)F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by (19)F NMR.

Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes.

Oms66 is a Borrelia burgdorferi outer membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection with B. burgdorferi (IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using "host-adapted" B. burgdorferi B31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.

Effect of staphylococcal delta-lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier transform infrared spectroscopic, and X-ray diffraction studies.

We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our 31P NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are studied, the isotropic component of the 31P NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase 31P NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic 31P NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D(2)O. However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.

Cell Membrane Orientation Visualized by Polarized Total Internal Reflection Fluorescence

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C 18-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.

Avian Adenovirus Induces Ion Channels in Model Bilayer Lipid Membranes

The action of duck egg drop syndrome 1976 (EDS-76) adenovirus on model bilayer lipid membranes (BLM) has been investigated on planar egg phosphatidylcholine membranes and small unilamellar vesicles. It was found that the adenovirus formed channels in planar BLM in a pH-dependent manner. The addition of EDS-76 to planar BLM at pH 5 induced voltage-independent channel activity of about 60 pS conductivity after a lag phase. At pH 3, EDS-76 induced irregular spikes of current across the planar BLM which disappeared after several minutes. The adenovirus also was able to induce pH-dependent leakage of calcein-loaded liposomes. EDS-76 did not induce channel activity in planar BLM or liposome leakage at neutral pH.

Asymmetric partitioning of a polymer into a curved membrane

The partitioning of a flexible model polypeptide into curved and flat lipid bilayer model membranes has been investigated using Monte Carlo methods. It is shown that a curved lipid membrane exhibits a gradient of lipid density fluctuations across the bilayer. The fluctuations increase continuously from the side of lower curvature (outer layer) to the side of higher curvature (inner layer). It is found that curvature promotes partitioning of the polymer into the outer layer of the membrane. Moreover, it is shown that in the case of the curved bilayer, the polymer moves spontaneously from the outer to inner layer of the membrane, where it is subsequently localized. It is argued that the curvature-induced gradients of the fluctuations of the mean local lipid density and the local mean lipid orientation across the membrane is the entropical driving force for the observed translocation process.

A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout

A protein with an apparent M(r) of 28,000 was isolated from outer membrane preparations of Aeromonas salmonicida A440. The protein was tested for the ability to form pores, using a planar lipid bilayer model membrane system. The protein appeared to be a monomer with a single-channel conductance in 1.0 M KCl of 1.96 nS and a cation/anion permeability ratio of 2.91 +/- 0.68. These data show that the porin channel is comparable in size to OmpC and OmpF of Escherichia coli and is relatively nonselective, having some preference for cations over anions. The porin was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a polyclonal antibody was raised. Immunoblot analysis showed that an immunologically cross-reactive protein was present in other Aeromonas strains but not in strains of Vibrio or Yersinia. The N-terminal amino acid sequence of the porin was determined and was found to show some homology to an Aeromonas hydrophila outer membrane protein. This is the second porin species of A. salmonicida to be described, and it differs from the other in subunit molecular weight, aggregation properties, peptidoglycan association, pore size, and antigenicity. Rainbow trout (Oncorhynchus mykiss) immunized intraperitoneally with the purified porin protein were significantly protected from experimental A. salmonicida challenge. This is the first report of successful vaccination against A. salmonicida with a purified outer membrane component.