A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids.

The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and β-casein with phospholipids

The conformations adopted by β-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being α-helical and the β-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the β-casein is in a compressed state whereas the apoprotein is extended as α-helices in the plane of the interface. The chain-length dependences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 microM and 19 microM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 microM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other "single-chain" lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Effect of doxorubicin on the order of the acyl chains of anionic and zwitterionic phospholipids in liquid-crystalline mixed model membranes: absence of drug-induced segregation of lipids into extended domains.

We investigated the effect of the antineoplastic drug doxorubicin on the order of the acyl chains in liquid-crystalline mixed bilayers consisting of dioleoylphosphatidylserine (DOPS) or -phosphatidic acid (DOPA), and dioleoylphosphatidylcholine (DOPC) or -phosphatidylethanolamine (DOPE). Previous 2H-NMR studies on bilayers consisting of a single species of di[11,11-2H2]oleoyl-labeled phospholipid showed that doxorubicin does not affect the acyl chain order of pure zwitterionic phospholipid but dramatically decreases the order of anionic phospholipid [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 1096, 67-80]. In the present work, we studied mixed bilayers in which alternatively the anionic or the zwitterionic phospholipid component was 2H-labeled so as to monitor its individual acyl chain order. Doxorubicin decreased the order parameter of the mixed anionic and zwitterionic lipids by approximately the same amount and did not induce a clear segregation of the lipid components into extended, separate domains. The drug had a comparable disordering effect on mixed bilayers of unlabeled cardiolipin and 2H-labeled zwitterionic phospholipid, indicating the absence of extensive segregation also in that case. Upon addition of doxorubicin to bilayers consisting of 67 mol% DOPE and 33 mol% anionic phospholipid, a significant part of the lipid adopted the inverted hexagonal (HII) phase at 25 degrees C. This bilayer destabilization, which occurred only in mixtures of anionic phospholipid and sufficient amounts of DOPE, might be of physiological importance. Even upon formation of extended HII-phase domains, lipid segregation was not clearly detectable, since the relative distribution of 2H-labeled anionic phospholipid and [2H]DOPE between the bilayer phase and HII phase was very similar. Our findings argue against a role of extensive anionic/zwitterionic lipid segregation in the mechanism of action and toxicity of doxorubicin.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers

The association of bacterial lipopolysaccharide with artificial membranes was studied in an attempt to understand the mechanism of binding of lipopolysaccharide to cell surfaces and to look for an effect on membrane stability. The membrane models used were phospholipid bilayers and monolayers. As measured by survival time, lipopolysaccharide was found to decrease the stability of bilayers at a concentration of 300 μg/ml. When assayed by dielectric breakdown, an effect of lipopolysaccharide was noticeable at concentrations of 50 μg/ml. In studies involving the penetration of monomolecular films of various phospholipids, native and alkali-treated lipopolysaccharide both caused increases in surface pressure, and therefore penetrated the films. However, alkali-treated lipopolysaccharide was at least ten times more efficient than the native product in penetration. Alkali-treated lipopolysaccharide had a greater degree of surface activity than native lipopolysaccharide, since alkali-treated lipopolysaccharide formed monomolecular films by itself, whereas native lipopolysaccharide did not. The changes in the surface pressure and surface potential of phospholipid films produced by lipopolysaccharide in the subsolution suggested that the interaction of lipopolysaccharide with phospholipid monolayers was by a combination of penetration and adsorption to the undersurface.     

Interaction of LL-37 with model membrane systems of different complexity: influence of the lipid matrix

As the main difference between bacterial and mammalian cell membranes is their net charge, the focal point of consideration in many model membrane experiments with antimicrobial peptides is lipid headgroup charge. We studied the interaction of the human multifunctional peptide LL-37 with single phospholipid monolayers, bilayers, and bilayers composed of binary mixtures of the four phospholipid species predominantly used in model membrane experiments (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine). We found that 1), the effects on single lipid monolayers are not comparable to those on the corresponding bilayers; 2), there are four different effects of LL-37 on bilayers of the four lipids; 3), the preference of LL-37 for the specific lipids is roughly inversely related to chain packing density; and 4), in the binary lipid mixtures, one lipid—and not necessarily the charged one—generally governs the mode of lipid/peptide interaction. Thus, our results show that lipid net charge is not the decisive factor determining the membrane-perturbing mechanism of LL-37, but only one of several parameters, among them packing density, the ability to form intermolecular H-bonds, and lipid molecular shape, which emphasizes how profoundly the choice of the model system can influence the outcome of a study of lipid/peptide interaction.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 μM and 19 μM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 μM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (< 5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other “single-chain” lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.     

Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes.

Defensin A is an inducible cationic protein secreted in the hemolymph of fleshfly Phormia terranovae larvae in response to bacterial or septic injuries. Defensin A is known to permeabilize the bacteria cell membranes by forming voltage-dependent channels. The penetration of this small protein into lipid monolayers was studied as a function of the polar head and acyl chain length of phospholipids. The extent of penetration by defensin A is higher in monolayers made of anionic phospholipids than in monolayers made of zwitterionic phospholipids (phosphatidylcholines), because of electrostatic interactions. From the analysis of the compression isotherm parameters of mixed defensin A/phospholipid monolayers, it appears that defensin A interacts with phospholipid by forming 1:4 complexes. These complexes are not miscible in the lipid phase and induce microheterogeneity in the lipid membrane. These clusters might be related to the ion-channel structures responsible for the biological activity of defensin A.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05-0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [13C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the 13C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (P beta') phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

Thermodynamic and mechanical properties of model mitochondrial membranes

Cardiolipin is a unique four-tailed, doubly negatively charged lipid found predominantly within the inner mitochondrial membrane, and is thought to be influential in determining membrane potential and permeability. To determine the role of cardiolipin in modulating the properties of membranes, this study investigates the thermodynamics of mixed cardiolipin and phosphatidylcholine monolayers and bilayers. Gibbs free energy analysis of mixed monolayers indicates that at low cardiolipin concentrations (5-10 mol%), there is a positive deviation from ideality on a pure water subphase, while at physiological salt concentrations a negative deviation from ideality is observed. The mechanical properties of bilayers containing cardiolipin were measured using micropipette aspiration. Both apparent area compressibility modulus, as well as lysis tension, decrease with increasing cardiolipin content. This destabilization indicates a decrease in the cohesive energy of the membrane. This interplay between interactions of lipids in monolayers and bilayers, suggests cardiolipin plays a dual role in modulating membrane properties. Cardiolipin enhances lateral interactions between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentrations. Taken together, these findings correlate with the decreased permeability and creation of folds in the inner mitochondrial membrane.     

Interactions of the antimicrobial peptides temporins with model biomembranes. Comparison of temporins B and L

Temporins are short (10-13 amino acids) and linear antimicrobial peptides first isolated from the skin of the European red frog, Rana temporaria, and are effective against Gram-positive bacteria and Candida albicans. To get insight into their mechanism(s) of action, we compared the effects on model membranes exerted by two members of this family, viz., temporin B (LLPIVGNLLKSLL-NH(2)) and temporin L (FVQWFSKFLGRIL-NH(2)). More specifically, we measured their insertion into lipid monolayers as well as their effects on the structural dynamics of liposomal bilayers as revealed by diphenylhexatriene (DPH)- and pyrene-labeled phospholipids. We also observed the impact of these peptides on the topology of giant vesicles. Both temporins readily penetrate into lipid monolayers, their intercalation being enhanced in the presence of the common bacterial negatively charged phospholipid phosphatidylglycerol. Instead, the eukaryotic lipid cholesterol did to some extent counteract their penetration into the lipid films. Both temporin B and temporin L caused an enrichment of phospholipids in the bilayers, and in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), these peptides increased acyl chain order. Temporin B had practically no effect on giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), whereas rapid vesiculation was observed when POPG was present. In contrast, temporin L induced vesiculation of both SOPC and SOPC/POPG giant vesicles while the presence of cholesterol in SOPC giant vesicles attenuated this effect.     

Exploring the effect of cholesterol in lipid bilayer membrane on the melittin penetration mechanism

A vascular mimetic membrane system was used to investigate the effect of cholesterol content in lipid bilayer on the dynamics of the melittin-membrane penetration reaction with real-time monitoring by a piezoelectric sensor and the assessment morphology using atomic force microscopy (AFM). In the presence of 30% cholesterol in a noncharged phosphatidylcholine (PC) phospholipid membrane, KA1 (binding affinity constant) and KA2 (insertion affinity constant) derived from a two-step model decreased significantly. This result suggests that the high dose of cholesterol in phospholipid membrane inhibits both the binding and the insertion of melittin. Next, dynamic laser scattering and AFM were used to verify the structural changes of lipid bilayers in solutions and interfaces, respectively. The superstructures in both 0 and 10% cholesterol lipid bilayers were disrupted with penetration of melittin according to these verifications. However, kinetic analysis reveals that the different mechanisms are dependent on cholesterol, particularly for the insertion step.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05–0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [¹³C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the ¹³C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (Pβ′) phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

In vitro evaluation of the antioxidant activity and biomembrane interaction of the lazaroid U-74389G

The aim of this paper was to clarify whether the interaction of the lazaroid U-74389G with phospholipid membranes might be relevant as to its antioxidant activity. Thus we evaluated the "in vitro" antioxidant activity of U-74389G in two experimental models: 1) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical; 2) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidino-propane) hydrochloride, on mixed dipalmitoyl-phosphatidylcholine/linoleic acid unilamellar vesicles. Moreover, given that biophysical techniques may help in explaining the role of a drug in its interaction with the microenvironment of the model lipid membranes, we used a classical approach to investigate the U-74389G/model membrane interaction: the differential scanning calorimetry technique on dimyristoylphosphatidylcholine multilamellar and unilamellar vesicles and the Langmuir-Blodgett technique on dimyristoylphosphatidylcholine monolayers. The results evidenced the strong antioxidant activity of U-74389G (especially in a membranous system) and its capability to interact with and be transported across model membranes. Thus one can speculate that U-74389G can act as scavenger of chain-propagating lipid peroxyl radicals within the membranes and may be able to protect not only cell membranes, but also intracellular components against peroxidative attack. Furthermore, also if there is no certain proof that the effect on the lipid packing order may play a key role in its antioxidant activity, the fluidifying effect on phospholipid bilayers of U-74389G favourably complements its free radical scavenging characteristics.     

Membrane interactions and metal ion effects on bilayer permeation of the lipophilic ion modulator DP-109

DP-109, a lipophilic bivalent metal ion modulator currently under preclinical development for neurodegenerative disorders, was designed to have membrane-associated activity, thereby restricting its action to the vicinity of cell membranes. We describe the application of a colorimetric phospholipid/polydiacetylene (PDA) biomimetic membrane assay in elucidating DP-109 membrane interactions and penetration into lipid bilayers. In this membrane model, visible quantifiable color changes were monitored in studying membrane interactions. The colorimetric data identified a biphasic concentration-dependent interaction, with a break point around the critical micelle concentration (CMC) of DP-109. The kinetics and colorimetric dose-response profile of DP-109 indicate that the compound inserts into the lipid bilayers rather than being localized at the bilayer surface. Analysis of interactions of DP-109 with phospholipid/PDA vesicles in which ionic gradients were imposed indicates that membrane activity of DP-109 is strongly affected by electrochemical gradients imposed by K+ and Zn2+. The ionic gradient effects suggest that the insertion of DP-109 into the membrane may depend on the membrane potential.