Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene CH stretching mode the phase transition of the hydrocarbon chains near 40°C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

The effect of gramicidin A on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines with different chains lengths.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.     

Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine

Solid gramicidin A and S and their interaction with DPPC bilayers were examined by 2H NMR as well as 31P NMR and differential scanning calorimetry (DSC). The deuterium spectra arose from deuterons associated with the peptide through chemical exchange in 2H2O. The spectra from both peptides were characterized by a quadrupolar splitting parameter, omega Q/2 pi approximately 150 kHz, and an asymmetry parameter, eta approximately 0.17. An additional 33 kHz, eta = 0 component arising from deuterons on mobile ornithine side chains was present in gramicidin S. In the gel phase of dipalmitoylphosphatidylcholine liposomes the gramicidins gave spectra that had components identical with those obtained from the solids. In the liquid-crystalline phase gramicidin A containing samples gave multicomponent spectra with a maximum quadrupolar splitting value of 133 kHz, eta = 0. A minimum in the T2e was observed, coinciding with the onset of the broadened phase transition measured by DSC and 31P NMR, due to the onset of axial rotation of the peptide in the bilayer. The different powder patterns in the liquid-crystalline spectra from gramicidin A probably arise from different amide sites along the transmembrane channel. The broad component of the 2H NMR spectra from gramicidin S in liposome preparations was not affected by the lipid-phase transition. The T2e was also constant over this temperature range. The results are consistent with a location of gramicidin S at the membrane surface.     

The structure and function of gramicidin A embedded in interdigitated bilayer

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

Thermodynamic, motional, and structural aspects of gramicidin-induced hexagonal HII phase formation in phosphatidylethanolamine

The effect of gramicidin incorporation on the thermodynamic properties of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) dispersions was investigated by differential scanning calorimetry. The results show that incorporation of gramicidin in PC systems results in a decrease of the energy content of the gel to liquid-crystalline phase transition. When incorporated in PE systems, however, the peptide does not affect the properties of the gel to liquid-crystalline phase transition with the exception that at high gramicidin concentrations the onset of the melting process is shifted to a slightly lower temperature. We therefore assume that in the lamellar gel state of PE aggregation of the peptide occurs. To get more insight into the nature of the gramicidin-PE interaction, we studied the motional and structural details of HII phase formation in gramicidin/PE systems with the use of 31P and 13C nuclear magnetic resonance (NMR) and small-angle X-ray diffraction. In agreement with earlier results [Van Echteld, C. J. A., Van Stigt, R., de Kruijff, B., Leunissen-Bijvelt, J., Verkleij, A. J., & De Gier, J. (1981) Biochim. Biophys. Acta 648, 287-291] it was shown that gramicidin incorporation lowers and broadens the bilayer to hexagonal HII phase transition in PE systems. 31P NMR chemical shift anisotropy (CSA) measurements indicated that a phase separation occurs between a gramicidin-poor lamellar phase and a gramicidin-rich HII phase. From combined CSA and spin-lattice relaxation time (T1) measurements it was suggested that in the HII phase gramicidin decreases the molecular order and increases the rate of motion of the phosphate moiety of PE. In addition, 13C NMR line width measurements indicated that the acyl chains are more disordered in the HII phase than in the lamellar phase and that a similar disorder occurs in the HII phase of the pure PE as in the gramicidin-rich HII phase. This interpretation was supported by the X-ray diffraction data, which show similar first-order repeat distances in both types of HII phase. From saturation-transfer NMR experiments in PE and gramicidin-PE mixtures it was shown that no exchange occurs between the lamellar and the HII phases in the time scale of 1-2 s, suggesting a macroscopic phase separation. Finally, we discussed the gramicidin-lipid interaction and in particular the HII phase formation by gramicidin in PE and in PC systems. It is proposed that aggregation of the peptide plays a crucial role in HII phase formation.     

The effects of gramicidin on the structure of phospholipid assemblies

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.     

Ultrasonic evidence for structural relaxation in large unilamellar liposomes

The ultrasonic absorption of large unilamellar vesicles (average diameter 0.2 micron) was determined in the frequency range 0.5-5 MHz. The liposomes were composed of a 4:1 mixture by weight of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol. They were studied with and without cholesterol or gramicidin incorporated into the bilayer. A large increase in absorption occurs at the solid to liquid-crystalline phase transition temperature (42 degrees C) of the pure lipid vesicles. This increase in absorption is interpreted as a structural relaxation of the 'melting' fatty acid chains occurring with an average relaxation time of 76 ns. The liposomes were also found to be extremely permeable near the transition temperature. Essentially complete release of cytosine arabinoside, a small water-soluble molecule, occurred at 42 degrees C. Addition of cholesterol or gramicidin to the bilayer of the liposomes broadened the ultrasonic absorption and reduced the efflux of cytosine arabinoside at the phase transition. No increase in absorption was observed at the transition temperature in the presence of 50 mol% of cholesterol. Gramicidin, in addition to broadening the transition, slows the isomerization of bonds in the hydrocarbon chains of the lipids. A concentration of 5 mol% gramicidin increased the average relaxation time to 211 ns.     

The influence of proteins and peptides on the phase properties of lipids

This paper reviews model membrane studies on the modulation of the macroscopic structure of lipids by lipid-protein interactions, with particular emphasis on the gramicidin molecule. This hydrophobic peptide has three main effects on lipid polymorphism: (1) in lysophosphatidylcholine it triggers a micellar to bilayer transition, (2) in phosphatidylethanolamine it lowers the bilayer to hexagonal H_II phase transition temperature and (3) in phosphatidylcholine and other bilayer preferring lipids it is able to induce the formation of an H_II phase. From experiments in which the gramicidin molecule was chemically modified it can be concluded that the tryptophan residues play a determining role in the peptide-induced changes in polymorphism. The experimental data lead to the proposal that gramicidin molecules have a tendency to self-associate, possibly mediated by tryptophan-tryptophan interactions and organize into tubular structures such as found in the H_II phase.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

Differential scanning calorimetry and 2H NMR studies of the phase behavior of gramicidin-phosphatidylcholine mixtures

The extents of two-phase coexistence in the phase diagrams of mixtures of gramicidin with 1,2-bis(perdeuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-d62) and with 1,2-bis(perdeuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d54) mixtures have been explored with differential scanning calorimetry (DSC) and deuterium nuclear magnetic resonance (2H NMR). For both systems, increased gramicidin content causes a decrease in transition enthalpy and a broadening of the peak in excess heat capacity at the transition. In DMPC-d54-based mixtures, the broadening is roughly symmetric about the pure lipid transition temperature. Addition of gramicidin to DPPC-d62 extends the excess heat capacity peak on the low-temperature side, resulting in a slightly asymmetric scan. Deuterium NMR spectra showing a superposition of gel and liquid-crystalline components, observed for both mixtures, indicate the presence of two-phase coexistence. For the DPPC-d62-based mixtures, two-phase coexistence is restricted to an approximately 2 degrees C temperature range below the pure transition temperature. For DMPC-d54-based mixtures, the region of two-phase coexistence is even narrower. For both mixtures, beyond a gramicidin mole fraction of 2%, distinct gel and liquid-crystal contributions to the spectra cannot be distinguished. Along with the broad featureless nature of the DSC scan in this region, this is taken to indicate that the transition has been replaced by a continuous phase change. These results are consistent with the existence of a closed two-phase region having a critical concentration of gramicidin below 2 mol%.     

A difference infrared spectroscopic study of gramicidin A, alamethicin and bacteriorhodopsin in perdeuterated dimyristoylphosphatidylcholine

Difference infrared spectroscopy has been used to study the way in which the intrinsic molecules gramicidin A, alamethicin and bacteriorhodopsin perturb their environment when present within a lipid bilayer structure. Dimyristoylphosphatidylcholine containing perdeuterated chains has been used to enable the lipid chain C-2H stretching absorption band to be separated from the C-H bands arising from the intrinsic polypeptide or protein. The C-2H stretching bands of the phospholipid are sensitive to two different types of chain conformation. The C-2H stretching frequency provides information about the static order of the lipid chains, whilst the half-maximum bandwidth provides a measure of chain librational and torsional motion. From the measurements it is concluded that: (1) Above the lipid phase transition temperature tc, low concentrations of either gramicidin A or alamethicin cause a small decrease in lipid chain gauche isomers whilst bacteriorhodopsin in the lipid bilayer has no effect. At higher concentrations each intrinsic molecule causes an increase to occur in lipid chain gauche isomers. (2) The lipid acyl chain motion, as deduced from the bandwidths is increased by the presence of a low concentration of gramicidin A within the lipid bilayer. The presence of the other intrinsic molecules studied have little effect. A higher concentration of alamethicin causes a decrease in chain motion whilst gramicidin A and bacteriorhodopsin have no effect. (3) Below tc each of the intrinsic molecules when present in the lipid bilayer causes an increase in gauche isomers to occur as well as an increase in the lipid chain motion. A broadening of the lipid phase transition occurs as the concentration of the polypeptide increases.     

Dielectric spectroscopy of L-alpha-lysolecithin-packaged gramicidin A

The complex permittivities of L-alpha-lysolecithin in the absence and presence of the gramicidin A ion channel were measured over the temperature range 0-60 degrees C and over the frequency range 1-1000 MHz. One dielectric relaxation/loss has been observed. It is located at 103.3 MHz (1.54 ns) for a micellar 0.4 M L-alpha-lysolecithin solution at 20 degrees C, whereas it is shifted to 71.7 MHz (2.22 ns) for a lamellar L-alpha-lysolecithin-gramicidin A aqueous solution (0.4 M L-alpha-lysolecithin, 0.0308 M gramicidin A) at 20 degrees C. The dielectric relaxation decreases and the relaxation time increases when gramicidin A is incorporated into L-alpha-lysolecithin. These dielectric changes are related, in part, to the micellar-to-lamellar lipid phase transition induced by the incorporation of gramicidin A into lysolecithin. We suggest that the diffuse rotational motion of the polar head group of L-alpha-lysolecithin contributes to the dielectric relaxation/loss at around 100 MHz.     

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl--phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.     

Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles

To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.     

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl–phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.     

Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

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.     

Interfacial properties of gramicidin and gramicidin-lipid mixtures measured with static and dynamic monolayer techniques.

Gramicidin films at the air/water interface are shown to exhibit a phase transition at 225 A2/molecule which might be caused by either cluster formation, reorientation of molecules, conformational changes or multilayer formation. It is further shown that coupling of a charged group on either NH2- or COOH-terminus or elongation of the peptide by two amino acids, only slightly affects the surface area characteristics whereas modification of the tryptophans or even replacement of a single tryptophan by phenylalanine leads to drastic alterations in the surface-area characteristics and a (partial) loss of the phase transition demonstrating that the tryptophans play an important role in the interfacial behavior of gramicidin. The lack of a solvent history effect on the interfacial behavior indicates a rapid conformational interconversion of the peptide at the air/water interface. Gramicidin in mixtures with dioleoylphosphatidylcholine and lysopalmitoylphosphatidylcholine shows a condensing effect whereas gramicidin shows ideal mixing with dioleoylphosphatidylethanolamine. The condensing effect most likely is related to the aggregational state of the peptides which is different in phosphatidylcholines and phosphatidylethanolamines.     

Phase separation and hexagonal HII phase formation by gramicidins A, B and C in dioleoylphosphatidylcholine model membranes. A study on the role of the tryptophan residues

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.