Conformational order of the hydrocarbon chains in lipid bilayers. A raman spectroscopic study

Raman spectra of dihexadecylphosphatidic acid (DHPA) and of dimyristoylphosphatidylcholine (DMPC) and its longer chain homologues have been obtained as a function of temperature in order to study the conformational order of the hydrocarbon chains in lipid bilayers. The frequency of the longitudinal acoustical (LA) vibration band is evaluated in terms of the length of all-trans chain segments. In the ordered phase, the chains are found to be overwhelmingly in the all-trans conformation. In the fluid phase, definite all-trans segments occur predominantly, the length of which coincides with the extension of the order parameter plateau known from deuterium magnetic resonance (DMR). The frequency of the skeletal optical (SO) trans vibration band leads to the same result, if evaluated under the assumption of vibrational decoupling by gauche bands in the fluid phase, thus lending support to this assumption. The intensity of this band determined from the band area increases linearly with chain length in the ordered phase and is independent of chain length in the fluid phase. Evaluating the intensity for the length of all-trans segments, the same result for the chain conformation is obtained as derived from the frequencies, with the additional information that the length of the all-trans segments in the fluid phase does not vary with chain length.     

Cholestryl-phosphoryl-choline in lipid bilayers.

Cholesteryl-phosphoryl-choline (CPC), a hybrid between cholesterol and lecithin, is incorporated into sonicated liposomes and erythrocyte membranes similarly to cholesterol. The effect of CPC on lipid microviscosity and degree of order is smaller, but not significantly than that of cholesterol. It is proposed that CPC may be employed as an efficient modulator of lipid dynamics.     

Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains

Quantitative structures are obtained at 30 degrees C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(qz) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides /F(qz)/ for low q(z). (3) Diffuse X-ray scattering from oriented stacks of bilayers provides /F(qz)/ for high q(z). Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 +/- 0.5 A(2) agrees well with our earlier publications, and we find A = 69.3 +/- 0.5 A2 for di22:1PC and A = 68.3 +/- 1.5 A2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.     

Helix-helix interactions in lipid bilayers.

Using a continuum model, we calculated the electrostatic interaction free energy between two alpha-helices in three environments: the aqueous phase, a low dielectric alkane phase, and a simple representation of a lipid bilayer. As was found in previous work, helix-helix interactions in the aqueous phase are quite weak, because of solvent screening, and slightly repulsive, because of desolvation effects that accompany helix assembly. In contrast, the interactions can be quite strong in a hypothetical alkane phase because desolvation effects are essentially nonexistent and because helix-helix interactions are not well screened. In this type of environment, the antiparallel helix orientation is strongly favored over the parallel orientation. In previous work we found that the free energy penalty associated with burying helix termini in a bilayer is quite high, which is why the termini tend to protrude into the solvent. Under these conditions the electrostatic interaction is strongly screened by solvent; indeed, it is sufficient for the termini to protrude a few angstroms from the two surfaces of the bilayer for their interaction to diminish almost completely. The effect is consistent with the classical model of the helix dipole in which the dipole moment is represented by point charges located at either terminus. Our results suggest, in agreement with previous models, that there is no significant nonspecific driving force for helix aggregation and, hence, that membrane protein folding must be driven by specific interactions such as close packing and salt-bridge and hydrogen bond formation.     

Energy transfer in lipid bilayers.

The quenching of fluorescence due to energy transfer between a dilute, random array of donor and acceptor chromophores in lipid bilayer was measured and compared to theoretical expressions developed to predict the decrease in emission intensity under these circumstances. The observed intensity was found to be the same function of quencher concentration in both planar, multilamellar dispersions and small, spherical vesicles. The degree of quenching was accurately predicted by a simple relation derived in this paper, as well as a more complex equation previously developed by Tweet, et al. The results suggest that significant quenching may be observed even when the average donor-acceptor separation exceeds the F?rster critical distance by severalfold. Application of these results to problems of current interest in membrane research are discussed.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.     

Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers.

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.     

Alamethicin-induced current-voltage curve asymmetry in lipid bilayers.

We have examined the causes of the asymmetry of the current-voltage curve induced by addition of alamethicin to one side of a black lipid membrane. We find that the alamethicin-induced current-voltage (I-V) curve has an inherent asymmetry. If it were possible to confine all alamethicin molecules to one side of the membrane, the I-V curve would exhibit a positive branch (voltage being measured with respect to the side of the membrane trans to the alamethicin addition) of steeper logarithmic slope than the negative branch and at a lower absolute value of potential. This condition is not usually realized, however, because alamethicin can leak through the membrane, so that, except at very high alamethicin concentrations and in certain kinds of membranes, the positive branch of the current-voltage curve has the same logarithmic slope as the negative branch and appears to arise from alamethicin which diffuses from the cis to the trans side of the membrane. We develop simple quantitative models for these two cases.     

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains.

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.     

Structural studies of polymer-cushioned lipid bilayers.

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.     

The sting. Melittin forms channels in lipid bilayers

Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance.     

Voltage-dependent capacitance in lipid bilayers made from monolayers.

Electrocompression has been measured in lipid bilayers made by apposition of two monolayers. The capacitance C(V), as a function of membrane potential, V, was found to be well described by C(V) = C(O) [1 + alpha(V + delta psi)2] where C(O) is the capacitance at V = O, alpha is the fractional increase in capacitance per square volt, and delta psi is the surface potential difference. In lipid bilayers made from monolayers alpha has a value of 0.02 V-2, which is ca. 500-fold smaller than the value found in solvent containing membranes. In asymmetric bilayers made of one neutral and one negatively charged monolayer, delta psi values were found to be those expected from independent measurements of surface charge density. If the fractional increase in capacitance found here is a good approximation to that of biological membranes, nonlinear capacitative charge displacement derived from electrostriction is expected to be less than 1% of the total gating charge displacement found in squid axons.     

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.     

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.     

"Reversed" alamethicin conductance in lipid bilayers.

Alamethicin at a concentration of 2 micrograms/ml on one side of a lipid bilayer, formed at the tip of a patch clamp pipette from diphytanoyl phosphatidylcholine and cholesterol (2:1 mol ratio) in aqueous 0.5 M KCl, 5 mM Hepes, pH 7.0, exhibits an asymmetric current-voltage curve, only yielding alamethicin currents when the side to which the peptide has been added is made positive. Below room temperature, however, single alamethicin channels created in such membranes sometimes survive a sudden reversal of the polarity. These "reversed" channels are distinct from transiently observed states displayed as the channel closes after a polarity reversal. Such "reversed" channels can be monitored for periods up to several minutes, during which time we have observed them to fluctuate through more than 20 discrete conductance states. They are convenient for the study of isolated ion-conducting alamethicin aggregates because, after voltage reversal, no subsequent incorporation of additional ion-conducting aggregates takes place.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Functional tethered lipid bilayers

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.     

Structure of lipid bilayers

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Polarity of lipid bilayers. A fluorescence investigation.

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)