gramicidin-a and 1-2-oleoylphosphatidylcholine

The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism. Recently, however, this mechanism was challenged, based on experiments with lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled. In these experiments, sustained channel activity was observed long after fusion had been terminated, which led to the proposal that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. This proposal is at odds with 40 years of experiments, but involves the key assumption that gA monomers do not exchange between bilayers. We tested the possibility of peptide exchange between bilayers using three different types of experiments. First, we demonstrated the exchange of gA between 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC

Topics: Bacterial Proteins; Brevibacillus; Electric Conductivity; Gramicidin; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Phosphatidylcholines; Spectrometry, Fluorescence; Unilamellar Liposomes

Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.

Topics: Arachidonic Acids; Cell Membrane; Endocannabinoids; Gramicidin; Lipid Bilayers; Membrane Proteins; Phosphatidylcholines; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X-100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X-100 occurs (Re (sat)), was determined by light scattering. Measurements of steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X-100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at Re (sat) was studied by CD spectroscopy. It was found that the conformational state of this channel-forming peptide changed crucially when Triton X-100 induced transition to more fluid membranes. The gA single-channel measurements were made with Triton X-100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid-detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane-active peptides.

Topics: Anti-Bacterial Agents; Cell Membrane; Detergents; Dynamic Light Scattering; Glycine max; Gramicidin; Liposomes; Membrane Fluidity; Membrane Potentials; Octoxynol; Phosphatidylcholines

A biomimetic membrane consisting of a thiolipid monolayer tethered to a mercury electrode, with a dioleoylphosphatidylcholine (DOPC) monolayer on top of it, was fabricated. The thiolipid, referred to as DPOL, consisted of an octaethyleneoxy (OEO) chain terminated at one end with a lipoic acid residue and covalently linked at the other end to two phytanyl chains. The functionality of this biomimetic membrane, referred to as a tethered bilayer lipid membrane (tBLM), was tested by incorporating gramicidin and alamethicin and verifying their ion channel activity. Advantages and drawbacks with respect to a tBLM using a thiolipid, referred to as DPTL, with a tetraethyleneoxy (TEO) chain were examined by using electrochemical impedance spectroscopy, potential-step chronocoulometry and cyclic voltammetry. The maximum charge surface density of potassium ions stored in the OEO spacer amounts to 70μCcm(-2), as compared to a charge surface density of 45μCcm(-2) in the TEO spacer. The lipid bilayer moiety of the DPOL/DOPC tBLM is somewhat leakier than that of the DPTL/DOPC tBLM at potentials negative of about -0.65V vs. the saturated calomel electrode. The estimated value of the surface dipole potential of the OEO spacer amounts to -0.180V and is, therefore, smaller than that, -0.230V, of the TEO spacer.

Topics: Biomimetic Materials; Cadmium; Cell Membrane; Dielectric Spectroscopy; Electrochemistry; Gramicidin; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Mercury; Phosphatidylcholines; Structure-Activity Relationship; Zinc

Gramicidin A (gA) dimers with bilayers, which consist of phospholipids and ionic liquids (ILs) at different molar ratios, were simulated at different salt concentrations of 0.15 and 1M NaCl. Bilayer thickness is larger than the length of a gA dimer, and hence lipids around the gA dimer are significantly disordered to adapt to the gA dimer, yielding membrane curvature. As the IL concentration increases, the bilayer thickness decreases and becomes closer to the gA length, leading to less membrane curvature. Also, ILs significantly increase lateral diffusivities of the gA dimer and lipids at 0.15M NaCl, but not at 1M NaCl because strong electrostatic interactions between salt ions and lipid head groups suppress an increase in the lateral mobility of the bilayer at high salt concentration. These findings help explain the conflicting experimental results that showed the increased ion permeability in electrophysiological experiments at 1M NaCl, but the reduced ion permeability in fluorescent experiments at 0.15M NaCl. ILs disorder lipids and make bilayers thinner, which yields less membrane curvature around the gA dimer and thus stabilizes the gA dimer, leading to the increased ion permeability. This IL effect predominantly occurs at 1M NaCl, where ILs only slightly increase the bilayer dynamics because of the strong electrostatic interactions between salt ions and lipids. In contrast, at 0.15M NaCl, ILs do not only stabilize the curved bilayer but also significantly increase the lateral mobility of gA dimers and lipids, which can reduce gA-induced pore formation, leading to the decreased ion permeability.

Topics: Gramicidin; Hydrophobic and Hydrophilic Interactions; Ion Transport; Ionic Liquids; Lipid Bilayers; Molecular Dynamics Simulation; Oligopeptides; Permeability; Phosphatidylcholines; Protein Multimerization; Sodium Chloride; Static Electricity

The cyclic voltammetry behavior of a mercury-supported tethered bilayer lipid membrane (tBLM) incorporating gramicidin A was investigated in aqueous 0.1 M KCl at pH 6.8, 5.4 and 3. The distal leaflet of the lipid bilayer consisted of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid or a DOPC/cholesterol mixture. In passing from pH 6.8 to pH 3, the midpoint potential between the negative current peak, due to K(+) inflow into the spacer, and the positive current peak, due to K(+) ejection into the aqueous solution, shifts toward more positive potentials, while the separation between these two peaks decreases. This behavior is interpreted quantitatively on the basis of a model relying on tBLM structural features estimated independently in previous works. The only adjustable parameter is the rate constant for cation translocation across a potential energy barrier located in the hydrocarbon tail region. The behavior is ascribed to a dragging of the lipid headgroups adjacent to the gramicidin channel mouth toward the hydrocarbon tail region, with a resulting decrease in the negative charge of the DOPC phosphate group, or of the DOPS carboxyl group, with decreasing pH.

Topics: Electrochemistry; Gramicidin; Hydrogen-Ion Concentration; Ion Channels; Lipid Bilayers; Membrane Potentials; Mercury; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Water

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e6 amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.

Topics: Adsorption; Fatty Acids, Unsaturated; Gramicidin; Lipid Bilayers; Membrane Potentials; Oxidants; Oxidation-Reduction; Oxidative Stress; Phosphatidylcholines; Singlet Oxygen; tert-Butylhydroperoxide

Poly-unsaturated fatty acids (PUFAs) alter the function of many membrane proteins, whereas monounsatured fatty acids generally are inert. We previously showed that docosahexaenoic acid (DHA) at pH 7 decreases the bilayer stiffness, consistent with an amphiphile-induced increase in elasticity, but not with a negative change in curvature; oleic acid (OA) was inert (Bruno, Koeppe and Andersen, Proc. Natl. Acad. Sci., 2007, 104, 9638-9643). To further explore how PUFAs and other amphiphiles may alter lipid bilayer properties, and thus membrane protein function, we examined how changes in acyl chain unsaturation and head group charge and size alter bilayer properties, as sensed by bilayer-spanning gramicidin A (gA) channels of different lengths. Compared to DHA, the neutral DHA-methyl ester has reduced effects on bilayer properties and 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC) forms bilayers that are softer than dioleoylphosphatidylcholine (DOPC). The changes in channel function are larger for the short gA channels, indicating that changes in elasticity dominate over changes in curvature. We altered the fatty acid protonation by titration: docosahexaenoic acid (DHA) is more potent at pH 9 (relative to pH 7) and is inert at pH 4; OA, which was inert at pH 7, becomes a potent modifier of bilayer properties at pH 9. At both pH 7 and 9, DHA and OA produced larger changes in the lifetimes of the short gA channels, demonstrating that they increase lipid bilayer elasticity when deprotonated--though OA promotes the formation of inverted hexagonal phases at pH 7. The positively charged oleylamine (OAm), which has a small head-group and therefore should be a negative curvature promoter, inhibited gA channel function with similar reductions in the lifetimes of the short and long gA channels, indicating a curvature-dominated effect. Monitoring the single-channel conductance, we find that the negatively charged fatty acids increase the conductance by increasing the local negative charge around the channel, whereas the positively charged OAm has no effect. These results suggest that deprotonated fatty acids increase bilayer elasticity by reversibly adsorbing at the bilayer/solution interface.

Topics: Amino Acid Sequence; Docosahexaenoic Acids; Fluorescence; Gramicidin; Hydrogen-Ion Concentration; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphatidylcholines; Phospholipids

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.

Topics: Algorithms; Capsaicin; Chromans; Energy Transfer; Genistein; Gramicidin; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Kinetics; Lipid Bilayers; Membrane Lipids; Models, Chemical; Octoxynol; Phosphatidylcholines; Protein Folding; Rosiglitazone; Thiazolidinediones; Troglitazone

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length--as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

Topics: Algorithms; Capsaicin; Cell Line; Cell Membrane; Genistein; Gramicidin; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Isoflavones; Kinetics; Lipid Bilayers; Membrane Potentials; Membrane Proteins; Octoxynol; Phloretin; Phosphatidylcholines; Protein Conformation

We find that the sensitivity of gramicidin A channels to the anesthetic halothane is highly lipid dependent. Specifically, exposure of membranes made of lamellar DOPC to halothane in concentrations close to clinically relevant reduces channel lifetimes by 1 order of magnitude. At the same time, gramicidin channels in membranes of nonlamellar DOPE are affected little, if at all, by halothane. We attribute this difference in channel behavior to a difference in the stress of lipid packing into a planar lipid bilayer, wherein the higher stress of DOPE packing reduces the degree of halothane partitioning into the hydrophobic interior.

Topics: Anesthetics, Inhalation; Gramicidin; Halothane; Kinetics; Lipid Bilayers; Lipids; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Phosphatidylethanolamines

Artificial bilayer membranes provide a platform for bioelectronic devices based on their structural, sensing, and transport functions. In this letter, we report on the impedance response of an engineered membrane with a lower leaflet of octadecanethiol on gold and an outer leaflet of dioleoylphosphatidylcholine with the monomeric channel protein gramicidin. This hybrid bilayer exhibits an electrical response analogous to a solid-state diode: the admittance is very low (<10(-)(7) Omega(-)(1) cm(-)(2)) over a wide potential range but increases exponentially at negative potentials.

Topics: Gold; Gramicidin; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membranes, Artificial; Models, Molecular; Phosphatidylcholines; Sulfhydryl Compounds; Surface Properties

A gramicidin A derivative with a polyether linkage between both ethanolamine termini was synthesized and its ion channel properties were studied. The compound showed a duplication in the state of conductance for alkali cations in thick DOPC bilayer membranes, which is interpreted as the occurrence of twin-channels. In thinner DMPC membranes mono-channels were dominant. The influence of hydrophobic coupling on the mono channel/twin channel equilibrium is discussed.

Topics: Dimerization; Dimyristoylphosphatidylcholine; Electric Conductivity; Gramicidin; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Membrane Fluidity; Membrane Lipids; Membranes, Artificial; Models, Chemical; Models, Molecular; Phosphatidylcholines; Protein Conformation

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air-water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (H(II)) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and (31)P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the H(II) phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.

Topics: Adsorption; Air; Animals; Cattle; Gramicidin; Hydrophobic and Hydrophilic Interactions; Liposomes; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phospholipids; Proteins; Scattering, Radiation; Surface Properties; Water; X-Ray Diffraction; X-Rays

A biosensor where the sensing surface is a fluid dioleyl phosphatidylcholine monolayer (DOPC) deposited on a mercury drop was used. The lipid monolayer was held in 0.1 M NaCl and a concentration of gramicidin A in the range 0-12 nM was used. Electrochemical impedance spectroscopy in the frequency range 0.1-65 kHz was employed to investigate how the defect-free monolayer responds to interactions of gramicidin A in solution. The data was analyzed both with multivariate data analysis and classical electrochemical methods. The principal component analysis of the resulting impedance spectra gave a linear dependence on the concentration of gramicidin A. An increasing permittivity was observed in the low-frequency regime with increasing concentration of gramicidin A in solution.

Topics: Biosensing Techniques; Electric Capacitance; Electric Impedance; Electrochemistry; Electrodes; Gramicidin; Lipid Bilayers; Mercury; Multivariate Analysis; Phosphatidylcholines

Gramicidin is an antibiotic peptide that can be incorporated into the monolayers of cell membranes. Dimerization through hydrogen bonding between gramicidin monomers in opposing leaflets of the membrane results in the formation of an iontophoretic channel. Surrounding phospholipids influence the gating properties of this channel. Conversely, gramicidin incorporation has been shown to affect the structure of spontaneously formed lipid assemblies. Using small-angle x-ray diffraction and model systems composed of phospholipids and gramicidin, the effects produced by gramicidin on lipid layers were measured. These measurements explore how peptides are able to modulate the spontaneous curvature properties of phospholipid assemblies. The reverse hexagonal, H(II), phase formed by dioleoylphosphatidylethanolamine (DOPE) monolayers decreased in lattice dimension with increasing incorporation of gramicidin. This indicated that gramicidin itself was adding negative curvature to the lipid layers. In this system, gramicidin was measured to have an apparent intrinsic radius of curvature, R0pgram, of -7.1 A. The addition of up to 4 mol% gramicidin in DOPE did not result in the monolayers becoming stiffer, as measured by the monolayer bending moduli. Dioleoylphosphatidylcholine (DOPC) alone forms the lamellar (L(alpha)) phase when hydrated, but undergoes a transition into the reverse hexagonal (H(II)) phase when mixed with gramicidin. The lattice dimension decreases systematically with increased gramicidin content. Again, this indicated that gramicidin was adding negative curvature to the lipid monolayers but the mixture behaved structurally much less consistently than DOPE/gramicidin. Only at 12 mol% gramicidin in dioleoylphosphatidylcholine could an apparent radius of intrinsic curvature of gramicidin (R0pgram) be estimated as -7.4 A. This mixture formed monolayers that were very resistant to bending, with a measured bending modulus of 115 kT.

Topics: Anti-Bacterial Agents; Biophysical Phenomena; Biophysics; Dimerization; Gramicidin; Hydrogen Bonding; Lipid Bilayers; Models, Statistical; Osmosis; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Temperature; Water; X-Ray Diffraction

The potential independent limiting flux of hydrated Tl(+) ions through gramicidin (GR) channels incorporated in phospholipid monolayers self assembled on a hanging mercury-drop electrode is shown to be controlled both by diffusion and by a dehydration step. Conversely, the potential independent limiting flux of dehydrated Tl(+) ions stemming from Tl amalgam electro-oxidation is exclusively controlled by diffusion of thallium atoms within the amalgam. Modulating the charge on the polar heads of dioleoylphosphatidylserine (DOPS) by changing pH affects the limiting flux of hydrated Tl(+) ions to a notable extent, primarily by electrostatic interactions. The dipole potential of DOPS and dioleoylphosphatidylcholine (DOPC), positive toward the hydrocarbon tails, does not hinder the translocation step of Tl(+) ions to such an extent as to make it rate limiting. Consequently, incorporation in the lipid monolayer of phloretin, which decreases such a positive dipole potential, does not affect the kinetics of Tl(+) flux through GR channels. In contrast, the increase in the positive dipole potential produced by the incorporation of ketocholestanol causes the translocation step to contribute to the rate of the overall process. A model providing a quantitative interpretation of the kinetics of diffusion, dehydration-hydration, translocation, and charge transfer of the Tl(+)/Tl(0)(Hg) couple through GC channels incorporated in mercury-supported phospholipid monolayers is provided. A cut-off disk model yielding the profile of the local electrostatic potential created by an array of oriented dipoles located in the lipid monolayer along the axis of a cylindrical ion channel is developed.

Topics: Anti-Bacterial Agents; Cell Membrane; Gramicidin; Hydrogen-Ion Concentration; Ions; Ketocholesterols; Kinetics; Lipids; Mercury; Models, Chemical; Models, Statistical; Phosphatidylcholines; Phosphatidylserines; Protein Transport; Thallium; Thermodynamics; Time Factors

Potential step amperometry (chronoamperometry) of the Tl(I)/Tl(Hg) electrochemical reduction process has been used to investigate the underlying mechanisms of gramicidin activity in phospholipid monolayers. The experiments were carried out at gramicidin-modified dioleoyl phosphatidylcholine (DOPC)-coated electrodes. Application of a potential step to the coated electrode system results in a current transient that can be divided into two regions. An initial exponential decay of current corresponds to the inactivation of monomer channel conductance and a longer time scale quasi-steady-state represents the diffusion of ions to a bimolecular surface reaction. Concentrations of monomer conducting channels are relatively low, and the results indicate that two or more forms of gramicidin are in equilibrium with each other in the layer. Aromatic/conjugated compounds incorporated into the monolayer increase the reduction current by decreasing the rate of channel inactivation and increasing the stability of the conducting channel. This effect is positively correlated with the degree of the compound's aromaticity. The anomalous influence of alkali metal ions on the reduction current is consistent with the model of gramicidin being speciated in the monolayer in more than one form. The results have implications on the lability of the peptide conformation in biological membranes and its dependence on lipid environment, solution composition, and applied potential.

Topics: Electric Conductivity; Electrochemistry; Electrodes; Electrolytes; Gramicidin; Ion Channels; Ion Transport; Kinetics; Membrane Potentials; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Solutions; Vitamin A

We have used magnetic alternating current mode atomic force microscopy (MAC-AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.

Topics: Anti-Bacterial Agents; Gramicidin; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phospholipids; Surface Properties; Time Factors

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Topics: Dimerization; Electrochemistry; Gramicidin; Ion Channels; Lipid Bilayers; Models, Biological; Models, Molecular; Patch-Clamp Techniques; Phosphatidylcholines; Probability; Stress, Mechanical; Thermodynamics

We have investigated the effect of gramicidin A on the dynamics of two model membranes: dimyristoylphosphatidylcholine (DMPC) in the lamellar phase at a lipid-to-peptide molar ratio of 10:1 and dioleoylphosphatidylcholine (DOPC) in the hexagonal HII phase at a lipid-to-peptide molar ratio of 5:1. Natural abundance 13C nuclear magnetic resonance (NMR) spectroscopy was used in combination with magic angle spinning to increase the spectral resolution, therefore allowing the different regions of the lipid bilayers to be investigated from the same spectra. 31P NMR was also used to detect and confirm the formation of the DOPC HII phase in the presence of gramicidin A. In order to examine the effect of gramicidin A on both the fast and slow motions of DMPC and DOPC, the 1H spin-lattice relaxation times in the laboratory frame (HT1) as well as the 1H spin-lattice relaxation times in the rotating frame (HT1rho) were calculated for each resolved protonated lipid resonance in the 13C spectra. For both DMPC and DOPC, we found that the presence of gramicidin A does not significantly affect the fast motions of the lipid acyl chains but increases slightly the fast motions of the polar head group. However, the HT1rho are significantly decreased, this effect being more pronounced for DOPC most likely due to a decrease in the rate of the lipid lateral diffusion.

Topics: Carbon Isotopes; Dimyristoylphosphatidylcholine; Gramicidin; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines

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.

Topics: Alkanes; Amino Acid Sequence; Fatty Acids, Unsaturated; Gramicidin; Ion Channels; Lipid Bilayers; Models, Biological; Models, Structural; Molecular Sequence Data; Phosphatidylcholines; Protein Structure, Secondary; Structure-Activity Relationship

Gramicidin derivatives with (positively-charged) C-terminal amino groups are found to incorporate readily and refold quickly when added to dioleoylphosphatidylcholine lipid vesicles from concentrated methanol solutions. Neutral and negatively-charged derivatives do not.

Topics: Circular Dichroism; Gramicidin; Phosphatidylcholines; Protein Folding; Structure-Activity Relationship

We investigated the effects of the hydrophobic small peptide antibiotic gramicidin A (gA) on the properties of vesicle bilayers in the liquid crystalline state. Time-resolved fluorescence anisotropy experiments were performed with unilamellar vesicles of the lipids DMPC, POPC, DOPC, EGGPC, DLPC, DOPG, and SQDG containing various concentrations of gA in two different conformations using TMA-DPH and DPHPC as fluorescent probes. These analogues of DPH were taken to study the gA induced change in the structural and dynamical properties of the lipid bilayer in different portions of the hydrophobic region. The time-resolved anisotropy data were analyzed using the recently introduced compound motion model [van der Sijs, D. A., et al. (1993) Chem. Phys. Lett. 216, 559; Muller, J. M., et al. (1994) Chem. Phys. 185, 393]. In general, gA raises the order and reduces the rotational diffusion coefficient for the probes in the bilayer. In DOPC vesicles this ordering effect of gA on the bilayer is found to depend on both the conformation of the peptide and the depth in the bilayer at which the order is probed. This significant effect of gA conformation on the lipid order parameter profile suggests that the shape of the gA dimer in the bilayer, which is determined by its conformation, affects the order of the adjacent DOPC lipid acyl chains.

Topics: Diphenylhexatriene; Fluorescence Polarization; Fluorescent Dyes; Gramicidin; Lipid Bilayers; Molecular Conformation; Molecular Probes; Molecular Structure; Phosphatidylcholines; Thermodynamics

Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, gramicidin M+ (gM+), as well as their enantiomers [gramicidin A- (gA-) and gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two gramicidin A enantiomers, or the two gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between gramicidin channels and the lipids in the host bilayer cannot be important determinants of gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.

Topics: Amino Acid Sequence; Electric Conductivity; Esters; Ethers; Gramicidin; Ion Channels; Lipid Bilayers; Molecular Sequence Data; Phosphatidylcholines; Stereoisomerism

The tryptophans in the gramicidin channel play a crucial role in the organization and function of the channel. The localization and dynamics of these tryptophans have been studied using fluorescence spectroscopy, especially utilizing environment-induced effects on the rates of solvent relaxation around these residues in membranes. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the tryptophans in the gramicidin channel exhibit a red edge excitation shift (REES) of 6 nm. In addition, fluorescence polarization shows both excitation and emission wavelength dependence. Fluorescence lifetime analysis shows a biexponential decay, corresponding to a short- and a long-lifetime component. The mean lifetime was found to be dependent on both excitation and emission wavelengths. Analysis of time-resolved emission spectra (TRES) shows a heterogeneous environment for the tryptophans consistent with the lifetime information. Taken together, these observations point out the motional restriction experienced by the tryptophans in the gramicidin channel. This is consistent with other studies in which such restrictions are thought to be imposed due to hydrogen bonding between the indole rings of the tryptophans and the neighboring lipid carbonyls. The significance of such organization in terms of functioning of the channel is brought out by the fact that substitution, photodamage, or chemical modification of these tryptophans is known to give rise to channels with conformation and reduced conductivity.

Topics: Gramicidin; Ion Channels; Lipid Bilayers; Mathematics; Models, Structural; Models, Theoretical; Phosphatidylcholines; Protein Conformation; Spectrometry, Fluorescence; Tryptophan

A controlled exchange of calcium between the extracellular space (mM Ca2+) and the neuroplasm (microM Ca2+) is considered to be an essential prerequisite for almost every stage of neuronal activity. Our research interest is focused on those compounds, which due to their physico-chemical properties and localization within the synaptic membrane might fulfill the task as neuromodulators for functional synaptic proteins. Because of this specific binding properties towards calcium and their peculiar interactions with calcium in model systems gangliosides (amphiphilic sialic acid containing glycosphingolipids) are favorite candidates for a functional involvement in synaptic transmission of information. In this study we used monolayers to investigate the molecular packing and surface potential at the air/water interface, the interaction of gangliosides with the depsipeptide valinomycin (= monovalent ion carrier), and its influenceability by calcium. Furthermore we looked at calcium effects on the single channel conductance and mean channel life-time of the monovalent ion channel gramicidin A in mixed PC/ganglioside bilayers. In pure ganglioside monolayers the addition of 0.01 mM Ca2+ induces monolayer condensation, a rise in collapse pressure (= higher film stability), a shift of phase transition (= change of conformation), and a more negative head group potential (change of electric properties). In mixed ganglioside-valinomycin monolayers the addition of Ca2+ causes phase separation and/or aggregate formation between the ganglioside and the peptide. Single channel conductance fluctuations as well as mean channel life-time were analyzed for gramicidin A incorporated into binary mixed black lipid membranes of negatively charged gangliosides (GM1, GD1a, GT1b, GMix) and neutral lecithin (DOPC) in different molar ratios. At monovalent electrolyte concentrations up to < 250 mM CsCl the single channel conductance was significantly larger in the negatively charged mixed DOPC/ganglioside membranes than in the neutral DOPC membrane. Additionally, in the presence of gangliosides the mean channel life-time is increased. The addition of calcium (0.05 mM) induced a reduction of single channel conductance of gramicidin A in DOPC- and mixed DOPC/ganglioside membranes. These physico-chemical data in connection with new electromicroscopical evidences for a precise localization of calcium, a calcium pump (Ca(2+)-ATPase), a clustered arrangement of gangliosides in synaptic termina

Topics: Animals; Calcium; Gangliosides; Gramicidin; Kinetics; Lipid Bilayers; Liposomes; Membrane Potentials; Models, Neurological; Neuronal Plasticity; Phosphatidylcholines; Phosphatidylserines; Pressure; Structure-Activity Relationship; Surface Properties; Synapses; Synaptic Membranes; Valinomycin

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.

Topics: Animals; Cell Membrane Permeability; Cholesterol; Diffusion; Electric Conductivity; Ergosterol; Erythrocyte Membrane; Gramicidin; Humans; Ion Channels; Lipid Bilayers; Phosphatidylcholines; Rubidium Radioisotopes; Tryptophan

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

Topics: Freeze Fracturing; Gramicidin; Liposomes; Membrane Fusion; Membrane Lipids; Microscopy, Electron; Molecular Conformation; Permeability; Phosphatidylcholines; Structure-Activity Relationship

With AMBER (assisted model building with energy refinement) molecular modelling techniques, the interactions between lipids which differ in the type of chain linkage (e.g., ether or ester) and gramicidin were approximated. It was found, theoretically, that replacement of the ester function in dipalmitoylphosphatidylcholine (DPPC) by an ether moiety induces a shift in the rotameric distribution of the Trp-15 side-chain in gramicidin A. Concomitantly, the channel entrance is contracted by approx. 0.4 A. The perturbation can be related to the strong hydrogen-bond formed between the lipid carbonyl group and the indole proton of the Trp-15 residue of gramicidin. In the ether lipid-gramicidin assembly a weaker H-bond is formed between Trp-15 and the phosphate moiety. To obtain a first indication of the influence of the strength of this H-bond on gramicidin A, a preliminary experimental study was set up. The transport properties of gramicidin A were studied using efflux measurements through vesicle walls containing ether and ester lipids, respectively. A change in the permeability of gramicidin A was found when ether lipids were added to a bilayer composed of the ester lipid dioleoylphosphatidylcholine (DOPC).

Topics: 1,2-Dipalmitoylphosphatidylcholine; Carboxylic Acids; Computer Simulation; Esters; Ethers; Gramicidin; Hydrogen Bonding; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Permeability; Phosphatidylcholines; Phospholipids; Protein Conformation; Sodium; Tryptophan

The behavior of two gramicidins incorporated into lipid monolayers is analyzed on the basis of the force and surface potential area curves. It is shown that the position of the gramicidins (helical axis parallel or perpendicular to the interface) depends on the monolayer pressure and that these molecules are not miscible with dioleoylphosphatidylcholine. Surface potential measurements suggest the existence of a relationship between the single channel characteristics and the surface potential and indicate that the tryptophans are essential for lowering the lipid surface potential in agreement with the single channel behaviour of both gramicidin A and gramicidin M.

Topics: Gramicidin; Ion Channels; Liposomes; Membrane Potentials; Models, Theoretical; Phosphatidylcholines; Pressure; Structure-Activity Relationship; Surface Properties

The role of the tryptophan-residues in gramicidin-induced HII phase formation was investigated in dioleoylphosphatidylcholine (DOPC) model membranes. 31P-NMR and small angle X-ray diffraction measurements showed, that gramicidin A and C (in which tryptophan-11 is replaced by tyrosine) induce a similar extent of HII phase formation, whereas for gramicidin B and synthetic analogs in which one tryptophan, either at position 9 or 11 is replaced by phenylalanine, a dramatic decrease of the HII phase inducing activity can be observed. Modification of all four tryptophans by means of formylation of the indole NH group leads to a complete block of HII phase formation. Sucrose density centrifugation experiments on the various peptide/lipid samples showed a quantitative incorporation of the peptide into the lipid. For all samples in a 1/10 molar ratio of peptide to lipid distinct bands were found, indicative of a phase separation. For the gramicidin A'/DOPC mixture these bands were analyzed and the macroscopic organization was determined by 31P-NMR and small-angle X-ray diffraction. The results demonstrate that a quantitative phase separation had occurred between a lamellar phase with a gramicidin/lipid ratio of 1/15 and a hexagonal HII phase, which is highly enriched in gramicidin. A study on the hydration properties of tryptophan-N-formylated gramicidin in mixtures with DOPC showed that this analog has a similar dehydrating effect on the lipid headgroup as the unmodified gramicidin. In addition both the hydration study and sucrose density centrifugation experiments showed that, like gramicidin also its analogs have a tendency to aggregate, but with differences in aggregation behaviour which seemed related to their HII phase inducing activity. It is proposed that the main driving force for HII phase formation is the tendency of gramicidin molecules to self-associate and organize into tubular structures such as found in the HII phase and that whether gramicidin (analogs) form these or other types of aggregates depends on their tertiary structure, which is determined by intra- as well as intermolecular aromatic-aromatic stacking interactions.

Topics: Gramicidin; Magnetic Resonance Spectroscopy; Membranes, Artificial; Phosphatidylcholines; Tryptophan; X-Ray Diffraction

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.

Topics: Deuterium; Gramicidin; Magnetic Resonance Spectroscopy; Models, Biological; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Thermodynamics

It is shown by 31P-NMR and small angle X-ray scattering that induction of an hexagonal HII phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to HII phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for HII phase formation by gramicidin.

Topics: Chemical Phenomena; Chemistry, Physical; Gramicidin; Magnetic Resonance Spectroscopy; Membranes, Artificial; Molecular Conformation; Phosphatidylcholines; Solvents; X-Ray Diffraction

It is shown that N-formylation of the tryptophan residues of gramicidin completely and reversibly blocks the hexagonal HII phase-inducing ability of the peptide in dioleoylphosphatidylcholine model membranes.

Topics: Amino Acid Sequence; Chemical Phenomena; Chemistry; Formates; Gramicidin; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Lipids; Phosphatidylcholines; Structure-Activity Relationship; Tryptophan

The macroscopic organization, lipid head group conformation, and structural and dynamic properties of 2H2O were investigated in dioleoylphosphatidylcholine (DOPC) model systems of varying gramicidin and 2H2O (or H2O) content by means of small-angle X-ray diffraction and 31P and 2H NMR. At low stages of hydration, N less than 6 (N = 2H2O/DOPC molar ratio), a single lamellar phase is observed in which the gramicidin molecules become preferentially hydrated upon increasing N. For 6 less than N less than 12 phase separation occurs between a gramicidin-poor and a gramicidin-rich lamellar phase. This latter phase is characterized by a smaller repeat distance and decreased DOPC head group order. For N greater than 12, the gramicidin-rich lamellar phase converts to a hexagonal HII phase. Thus, hydration of gramicidin is a prerequisite for HII phase formation in the DOPC/gramicidin system. The HII phase is very rich in gramicidin and 2H2O (gramicidin:DOPC:H2O = 1:1.1:0.9 w/w/w). A model is proposed in which self-assembly of hydrated gramicidin molecules into domains of a specific structure plays a determinant role in the formation of the HII phase by gramicidin.

Topics: Gramicidin; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Biological; Molecular Conformation; Phosphatidylcholines; Water; X-Ray Diffraction

We have analyzed the effects induced in different phospholipid planar bilayers by monosialoganglioside micelles containing the ionophore gramicidin D. The membrane conductance increases after the addition of GM1 micelles at various ionophore/ganglioside ratios. We believe this fact may be ascribed to gramicidin molecules that incorporate into the bilayer together with gangliosides. In the presence of micelles the mean lifetime and the amplitude of the gramicidin single channel did not present relevant modifications when dioleoylphosphatidylcholine or phosphatidylserine were used to form the bilayer. Calcium proved to trigger the interaction between phosphatidylethanolamine membranes and GM1 micelles containing gramicidin. In this case the ionic pore presents a longer lifetime and a lower amplitude with respect to pure gramicidin. We suggest that different properties developed by gramicidin may depend on structural organization of gangliosides when incorporated into the phospholipid bilayer.

Topics: Biophysical Phenomena; Biophysics; Calcium; Colloids; Electric Conductivity; G(M1) Ganglioside; Gangliosides; Gramicidin; Ion Channels; Lipid Bilayers; Micelles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines