Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.     

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.     

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%.     

The effect of changes in gramicidin conformation on bilayer lipid properties.

The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation.     

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.     

Solid-state NMR study of trehalose/1,2-dipalmitoyl-sn-phosphatidylcholine interactions

31P and 2H solid-state NMR studies of dry trehalose (TRE) and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) mixtures are reported. 31P spectra are consistent with a rigid head group above and below the calorimetric phase transition for both dry DPPC and a dry 2:1 TRE/DPPC mixture. In addition, 2H spectra of DPPC labeled at the 7-position of the sn-2 chain (2[7,7-2H2]DPPC) show exchange-narrowed line shapes with a width of 120 kHz over the temperature range 25-75 degrees C. These line shapes can be simulated with a model involving two-site jumps of the deuteron. In contrast, the 2H NMR spectrum of a dry 2:1 TRE/2[7,7-2H2]DPPC mixture above the phase transition (Tc = 46 degrees C) is narrowed by a factor of approximately 4 to a width of 29 kHz. Simulation of this spectrum requires a model involving four-site jumps of the deuteron and is indicative of highly disordered lipid acyl chains similar to those found in the L alpha-phases of hydrated lipids. Thus, TRE/DPPC mixtures above their transition temperatures exist in a new type of liquid crystalline like phase, which we term a lambda-phase. The observation of the dynamic properties of this new phase indicates the mechanism by which anhydrobiotic organisms maintain the integrity of their membranes upon dehydration.     

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.     

Deuterium magnetic resonance study of the gel and liquid crystalline phases of dipalmitoyl phosphatidylcholine.

Deuterium magnetic resonance is applied to the study of the liquid crystalline and gel phases, and of the phase transition, of a multilamellar dispersion of chain perdeuterated (d62)-dipalmitoyl phosphatidylcholine/H2O. Analysis of the deuterium spectra in terms of the moments of the spectra allows one to make quantitative statements concerning the distribution of quadrupolar splittings even in complicated situations, e.g., when using perdeuterated sampled or when there are mixed phases. This analysis indicates that d62-dipalmitoyl phosphatidylcholine in excess H2O undergoes a sharp phase transition (with a width of less than 1 degree C) at approximately 37 degrees C and that there appears to be hysteresis in the phase transition of approximately 1 degree C. In the lamellar liquid crystalline phase above 37 degrees C the spectra show a number of well-resolved features whose quadrupolar splittings can be followed as the temperature is varied. The gel phase near 20 degrees C possesses a very broad, almost featureless spectrum that does not seem to support a model of the gel phase wherein the hydrocarbon chains are fully extended in the all-trans conformation. At temperatures near 0 degrees C the spectra clearly indicate that a large fraction of the lipid molecules cease the rotation about their long axes, giving a spectrum more characteristic of a rigid or solid sample. These results give a picture of the gel phase as a phase characterized by considerable hydrocarbon chain disorder near 20 degrees C and becoming a more solid-like phase near 0 degrees C. The spin-lattice relaxation time, T1, has been measured at 20 degrees C in the gel phase, and at 37 and 45 degrees C in the liquid crystalline phase. The values of T1 obtained for each of the resolvable peaks in the spectrum at 37 degrees C are compared to the values (for each peak) of T2e, the decay time of the quadrupolar echo, obtained at the same temperature. These results are discussed in terms of a simple two-motion model.     

Gramicidin-induced hexagonal HII phase formation in erythrocyte membranes

Using 31P nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and freeze-fracture electron microscopic (FFEM) techniques, it is shown that gramicidin induces a hexagonal HII phase not only in liposomes prepared from total lipids extracted from human erythrocytes but also in isolated human erythrocyte membranes (white ghosts). A 37 degrees C, HII phase formation is detected at a gramicidin to phospholipid molar ratio exceeding 1:80. At a molar ratio of 1:5, about 30% of the phospholipid is organized in the HII phase. The gramicidin-induced HII phase exhibits a very small 31P chemical shift anisotropy [(CSA) approximately 10 +/- 1 ppm], indicating decreased head-group order, and it displays a temperature-dependent increase in tube diameter from 60.2 A at 4 degrees C to 64.2 A at 37 degrees C in ghosts and from 62.8 to 69.4 A at 37 degrees C in total lipid extracts, both in the presence of 1 mol of gramicidin/10 mol of phospholipid. This anomalous temperature-dependent behavior is probably due to the presence of cholesterol. 31P NMR data indicate that the HII phase formation by gramicidin is temperature dependent and show the gradual disappearance of the HII phase at low temperatures (less than 20 degrees C), resulting in a bilayer type of 31P NMR line shape at 4 degrees C, whereas SAXS and FFEM data suggest equal amounts of HII phases at all temperatures. This apparent discrepancy is probably the result of a decrease in the rate of lateral diffusion of the membrane phospholipids which leads to incomplete averaging of the 31P CSA in the HII phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dielectric relaxation spectroscopy on dimyristoylphosphatidylcholine-packaged gramicidin A

The complex permittivities of aqueous suspensions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and of DMPC packaged gramicidin A' (DMPC-GA) have been determined over the frequency range of 1 MHz to 1 GHz and the temperature range of 0-60 degrees C. A dielectric relaxation/loss has been observed at about 66 MHz for the DMPC suspension (30 degrees C) and at about 57 MHz for the DMPC-GA suspension (30 degrees C). This dielectric relaxation/loss has been attributed to the rotational mobility of the zwitterionic group of DMPC. The temperature dependence (from 60 degrees C to 0 degrees C) of this dispersion/absorption process of the DMPC suspension indicates a sharp reduction of the dielectric relaxation at about 20 degrees C. This dielectric change is related to the conversions of shape and structure of bilayer aggregates. This sharp reduction of the dielectric relaxation disappears or broadens when GA is incorporated into the DMPC aqueous suspension. The interpretation of these results is that the GA addition into the DMPC aqueous suspension induces a small decrease of the rotational mobility of the zwitterionic group above the lipid phase transition, and a small increase of the rotational mobility of the zwitterionic group below the lipid phase transition.     

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.     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.     

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation.

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.     

Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines

The bilayer to hexagonal phase transition temperatures of dielaidoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine are 65.6 and 71.4 degrees C, respectively. Using high-sensitivity differential scanning calorimetry, I have shown that these transition temperatures are extremely sensitive to the presence of small amounts of other lipid components. For example, at a mole fraction of only 0.01, dilinolenin lowers the bilayer to hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by 8.5 degrees C. Other diacylglycerols have similar effects on this transition temperature, although the degree of unsaturation of the acyl chains has some effect, with distearin being less potent. In comparison, the 20-carbon alkane eicosane lowers this transition temperature by 5 degrees C, while palmitoyl-lysolecithin raises it by 2.5 degrees C. Similar effects of these additives on the bilayer to to hexagonal phase transition temperature are observed with dielaidoylphosphatidylethanolamine. At these concentrations of additive, there is no effect on the gel-state to liquid-crystalline-state transition temperature. The observed shifts in the temperature of the bilayer to the hexagonal phase transition can be qualitatively interpreted in terms of the effects of these additives on the hydrophilic surface area and on the hydrophobic volume. Substances expanding the hydrophobic domain promote hexagonal phase formation and lower the bilayer to hexagonal phase transition temperature. The sensitivity of the bilayer to hexagonal phase transition temperature to the presence of additives is at least as great as that which has been observed for any other lipid phase transition.     

Transverse nuclear spin relaxation in phosphatidylcholine bilayers containing gramicidin.

Deuterium nuclear magnetic resonance has been used to study transverse relaxation in samples of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, perdeuterated and specifically deuterated at the alpha position of the chains, containing the polypeptide gramicidin at concentrations of 0, 1, and 4 mol%. For 4 mol% gramicidin, the bilayer is thought to undergo a continuous phase change rather than a phase transition proceeding via two phase coexistence. Information is obtained regarding lipid dynamics in the continuous phase change region of the phase diagram. In the presence of gramicidin, the transverse relaxation time measured by the quadrupole echo technique, T2e, passes through a minimum in the gel phase. The gramicidin concentration dependence of T2e suggests that the polypeptide reduces the temperature sensitivity of the correlation time responsible for the minimum. The polypeptide also increases the sensitivity of the first spectral moment, M1, to the quadrupole echo pulse separation. This behavior is attributed to a polypeptide-induced enhancement of the spread in T2e along the acyl chains. Quadrupole Carr-Purcell-Meiboom-Gill experiments are used to separate contributions to the observed behavior from fast and slow motions.     

NMR Studies of lipid lateral diffusion in the DMPC/gramicidin D/water system: peptide aggregation and obstruction effects

The PFG-NMR method has been used in macroscopically oriented bilayers to investigate the effect of the peptide gramicidin D on the lateral diffusion of dimyristoylphosphatidylcholine. By varying both the temperature (21-35 degrees C) and the gramicidin content (0-5 mol %) we have introduced solid obstacles into the lipid liquid crystalline bilayer. It was shown that the obstruction effect exerted by the peptide can be described with several different theoretical models, each based on different premises, and that the fit of the models to experimental data gave reasonable results. We found that each gramicidin molecule was surrounded by approximately one layer of bound lipids and that the obstruction from gel phase patches can be described as small solid obstacles. No evidence of linear aggregates of gramicidin, such as those reported by atomic force microscopy in the gel phase, was found.     

Evidence from deuterium nuclear magnetic resonance for the temperature-dependent reversible self-association of erythrocyte band 3 in dimyristoylphosphatidylcholine bilayers

Band 3, isolated from human erythrocytes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) deuterated in the terminal methyl groups of the choline head group. By use of Triton X-100 for selective extraction and purification of band 3 and then cholate for subsequent solubilization with the lipid, a number of reconstituted complexes were produced by exhaustive detergent dialysis with protein:lipid weight ratios of between 0.32:1 and 1.25:1. Electron micrographs of negatively stained complexes showed that this method produced large vesicles of greater than 300-nm diameter. Deuterium nuclear magnetic resonance (NMR) spectra from the choline methyl deuterons in bilayer lipid above the liquid-crystal-gel phase transition temperature were shown to change systematically with increasing concentrations of band 3 in the bilayers. The measured quadrupole splittings, taken as the separation of the turning points in the recorded spectra, decreased from a value of 1.28 kHz for pure lipid to 0.98 kHz for bilayers with a protein:lipid ratio of 1.25:1 at 26 degrees C. At 35 degrees C, a more pronounced decrease in the quadrupole splittings was measured. The data from the complexes with protein:lipid ratios up to 0.7:1 (w/w) obey the mathematical treatment for a rapid two-site exchange between lipids at the protein-lipid interface and the bulk lipid phase. The temperature dependence of the measured quadrupole splitting with respect to the protein:lipid ratio indicates that the amount of lipid at the protein-lipid interface increases with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pressure effects on the structure and phase behavior of DMPC-gramicidin lipid bilayers: a synchrotron SAXS and 2H-NMR spectroscopy study

The influence of Gramicidin D (GD) incorporation on the structure and phase behavior of aqueous dispersions of DMPC lipid bilayers has been studied using small-angle x-ray scattering (SAXS) and (2)H-NMR spectroscopy. The experiments covered a temperature range from -10 degrees C to 60 degrees C and a pressure range of 0.001-4 kbar. Pressure was used to be able to tune the lipid bilayer conformational order and phase state and because high pressure is an important feature of certain natural biotopes. The data show that, depending on the GD concentration, the structure of the temperature- and pressure-dependent lipid phases is significantly altered by the insertion of the polypeptide, and a p,T-phase diagram could be obtained for intermediate GD concentrations. Upon gramicidin insertion, a rather narrow fluid-gel coexistence regions is formed. Two gel phases are induced which are different from those of the pure lipid bilayer system and which separate at low temperatures/high pressures. For both the temperature- and pressure-induced fluid-to-gel transition, a similar pseudocritical transitional behavior is observed, which is even more pronounced upon incorporation of the peptide.     

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.