Importance of the tryptophans of gramicidin for its lipid structure modulating activity in lysophosphatidylcholine and phosphatidylethanolamine model membranes. A comparative study employing gramicidin analogs and a synthetic α-helical hydrophobic polypeptide

The importance of the tryptophan residues of gramicidin for the lipid structure modulating activity of this pentadecapeptide was investigated by studying the interaction of gramicidin analogs A, B, C (which have a tryptophan, phenylalanine and tyrosine in position 11, respectively) and tryptophan-N-formylated gramicidin (in which the four tryptophan residues have been formylated) with several phospholipid systems. In addition in α-helical model pentadecapeptide (P15) was studied to further test the specificity of the gramicidin-lipid interaction. DSC experiments showed that all the gramicidin analogs produced a significant decrease in the gel to liquid-crystalline transition enthalpy of dipalmitoylphosphatidylcholine. The P15 peptide was much less effective in this respect. In dielaidoylphosphatidylethanolamine the gel → liquid-crystalline transition enthalpy was much less affected by the incorporation of these molecules. In this lipid system tryptophan-N-formylated gramicidin was found to be the most ineffective. ³¹P-NMR and small angle X-ray diffraction experiments showed that the ability of the peptides to induce bilayer structures in palmitoyllysophosphatidylcholine and H_II phase promotion in dielaidoylphosphatidylethanolamine systems follows the order: gramicidin A′ (natural mixture) ≈gramicidin A > gramicidin B ≈ gramicidin C > tryptophan-N-formylated gramicidin > P15. These results support the hypothesis that the shape of gramicidin and its aggregational behaviour, in which the tryptophan residues play an essential role, are major determinants in the unique lipid structure modulating activity of gramicidin.     

The effect of gramicidin A on phospholipid bilayers

The helical polypeptide, gramicidin A has been widely studied as a model for the interactions of hydrophobic proteins with lipid bilayer membranes. Many reports are now available of the physical effects of mixing gramicidin A with phospholipid membranes, however, the interpretation of these data remains unclear. The purpose of this communication is to examine the controversial claim that high concentrations of gramicidin A cause disorder within the L phase of phosphatidylcholine-water dispersions. Solid-state nuclear magnetic resonance (NMR), density gradient and X-ray diffraction techniques are used to confirm the existence of such an effect and mechanisms are discussed which account for the known effects of gramicidin A on lipid bilayers.     

Intrinsic protein-lipid interactions. Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+-ATPase in biomembranes and reconstituted systems

Infrared spectroscopy has been applied to the study of a number of aqueous systems of model and natural biomembranes. The absorption bands arising from water and buffer solutions were eliminated by means of an infrared spectrometer data station. Spectra were examined using H₂O and ²H₂O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca²⁺-ATPase were examined. The infrared spectra of the reconstituted biomembranes were compared with those of the corresponding natural biomembranes, i.e. the purple membrane of Halobacterium halobium and also sarcoplasmic reticulum membranes, respectively.Changes in lipid chain conformation caused by the various intrinsic molecules incorporated within the model lipid bilayer structures were monitored by studying the shifts in frequency (cm^?1) of the CH₂ symmetric and asymmetric absorption bands arising from the lipid chains. The effect of gramicidin A and also the intrinsic proteins, as indicated by the shift of band frequencies, are quite different from that of cholesterol at temperatures above the main lipid transition temperature t_c. Cholesterol causes a reduction in gauche isomers which increases with concentration of cholesterol within the lipid bilayer. Whilst gramicidin A and the intrinsic proteins at low concentration cause a reduction of gauche isomers, at higher concentrations of these molecules, however, there is little difference in gauche isomer content when the intrinsic molecule is present compared with that of the fluid lipid alone. These results are considered and compared with previously published studies using deuterium nuclear magnetic resonance spectroscopy on similar model biomembrane systems. Below the lipid t_c value, all the intrinsic molecules produce an increase in gauche isomers presumably by disturbing the lipid chain packing in the crystalline lipid arrangement.Information about the polypeptide structure within gramicidin A. the reconstituted proteins and also the proteins in the natural biomembranes was obtained by examining the region of the infrared spectrum between 1600 and 1700 cm^?1 associated with the amide I and amide II bands. An examination of the infrared band frequencies of the different systems in this region leads to the conclusions: (1) that gramicidin A within a phospholipid bilayer structure probably has a single helix rather than a double helix structure; (2) that there are differences in band widths of the reconstituted Ca²⁺-ATPase and bacteriorhodopsin compared with the spectra of the corresponding sarcoplasmic reticulum and purple membrane; (3) different membrane proteins adopt different conformations as evinced by a comparison of the spectra of the sarcoplasmic reticulum and purple membrane; (4) the polypeptide arrangement in the purple membrane is mainly helical but the abnormal frequency of the amide I band suggests that some distortion of the helix occurs: and (5) the sarcoplasmic reticulum membrane contains unordered as well as α-helix polypeptide arrangements.     

Gramicidin induces the formation of non-bilayer structures in phosphatidylcholine dispersions in a fatty acid chain length dependent way

The hydrophobic peptide gramicidin is shown by ³¹P-NMR, freeze-fracture electron microscopy and small-angle X-ray diffraction, to induce a hexogonal H_II-phase lipid organization when incorporated in liquid crystalline saturated and unsaturated synthetic and natural phosphatidylcholines if the length of the fatty acids exceeds a 16 carbon atoms chain. The amount of hexagonally organized lipid increases with increasing fatty acid chain length. With phosphatidylcholines possessing shorter fatty acid chains, as well as with the longer phosphatidylcholines in the gel state, a lamellar organization results. Of the various possible models to explain the induction of the hexagonal H_II phase by gramicidin, one in which gramicidin dimers span adjacent cylinders of the hexagonal H_II phase seems most plausible. In phosphatidylcholines with intermediary chain lengths gramicidin induces intermediary structures, such as lipidic particles and possibly cubic phases. These experiments suggest that the balance between the length of the hydrophobic domain of a peptide and the membrane thickness is of critical importance for the structure of the membrane. In relation to this observation, the possible involvement of non-bilayer lipid structures in insertion and anchoring of membrane proteins is discussed.     

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen

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.     

Effect of gramicidin on percutaneous permeation of a model drug

This study investigated the enhancement effect of gramicidin, a cationic ionophore, on percutaneous absorption of a model drug, benzoic acid (BA), through rat abdominal skin. The mechanisms by which gramicidin increased skin permeability to BA were also investigated. Degree of hydration measured by the Karl Fisher method, the concentration gradient measured by cryostat analysis, and lipid concentration measured by the. Fiske-Subbarow method were evaluated and compared. The results showed that BA permeation profiles through rat abdominal skin followed dose- and volume-dependent patterns. The pretreatment of gramicidin increased the permeation rate of BA through rat abdominal skin compared with the untreated control (18.89 vs. 10.86 μ g/cm² hour). Change in skin permeation rate of BA after gramicidin pretreatment was closely correlated with the remaining skin water content. There were no significant differences in the amounts of phospholipid phosphorous between gramicidin pretreated and untreated skin. The enhancing effect of gramicidin on percutaneous absorption of a model drug is mainly a tributed to increasing the diffusivity in the hydration domain of the skin and rearranging the lipid bilayer in the stratum corneum.     

Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope.

The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure.     

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H₂O₂ led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.     

Solvent history dependence of gramicidin-lipid interactions: a Raman and infrared spectroscopic study.

We have investigated the interactions between gramicidin and a model membrane composed of one phospholipid, dimyristoylphosphatidylcholine, as a function of the cosolubilization solvent and incubation time used in the sample preparation. Three organic solvents have been used; trifluoroethanol, a mixture of methanol/chloroform (1:1 v/v), and ethanol. Using Fourier transform infrared spectroscopy, we have demonstrated that the conformation adopted by gramicidin in the membrane is dependent upon the cosolubilization solvent used, and, only with trifluoroethanol, it is possible to incorporate gramicidin entirely as a beta 6.3-helix. Moreover, Raman spectroscopy results indicate that the orientation of the tryptophan side chains in gramicidin and their interaction with the hydrocarbon chains and the carbonyl groups of the lipids are also dependent on the cosolubilization solvent. On the other hand, the effect of the incorporation of gramicidin on the thermotropism of the lipid bilayer was found to be dependent upon the conformation of gramicidin in the lipid bilayers.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of cholesterol and gramicidin A′ on the carbonyl groups of dimyristoylphosphatidylcholine dispersions

Proton-enhanced carbon-13 magnetic resonance measurements have been made of the natural abundance carbon-13 carbons in hydrated L_α phase dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) codispersed with cholesterol or with the polypeptide gramicidin A′. The carbonyl group spectrum consists of a superposition of two peaks derived from the two carbonyl sites within the lipid. In the L_α phase of DMPC both carbonyl sites contribute axially symmetric spectra, one with a chemical shift anisotropy of –29 ppm and the other with a chemical shift anisotropy of less than –5 ppm. The chemical shift anisotropy of the broader carbonyl resonance was found to increase with increasing cholesterol content. However, in DMPC dispersions with gramicidin A′, the chemical shift anisotropy of the broader carbonyl signal initially increased slightly from that of pure DMPC and then decreased with increasing concentrations of gramicidin A′. The width of the narrower spectral component was essentially unaltered by cholesterol or gramicidin A′. The presence of a narrow component at all concentrations of cholesterol or gramicidin A′ suggests that it is unlikely that any significant conformational changes have occurred at the carbonyl level of the bilayer. We propose that the major effect of cholesterol or gramicidin A′ is to alter the molecular order parameter, S_mol, which reflects the range of angles through which the local molecular long axis of the phospholipid is tumbling.     

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.     

Comparison of lipid/gramicidin dispersions and cocrystals by Raman scattering

Gramicidin crystals, dimyristoylphosphatidylcholine (DMPC)/gramicidin dispersions, and DMPC/gramicidin cocrystals were examined by Raman scattering to determine lipid/gramicidin stoichiometries and lipid organization. Calibrations of the choline (716-cm-1) and tryptophan (756-cm-1) peaks indicate that the cocrystals contain two lipids for each gramicidin monomer, a result confirmed by chemical analyses of washed crystals. In dispersions with high lipid/gramicidin ratios (e.g., 25:1), the lipid is ordered but becomes increasingly disordered as the gramicidin content is increased. Paradoxically, the DMPC/gramicidin cocrystals have highly ordered lipids that possibly contain no gauche bonds at all, despite their low lipid/gramicidin ratio. In addition, the polypeptide amide I peak position near 1670 cm-1 is found to be independent of the lipid/gramicidin ratio in the complexes and may indicate a beta-helix-type secondary structure at all ratios. However, the amide I peak broadens significantly at low lipid/gramicidin ratios and broadens still further in the cocrystals, suggesting that protein-protein interactions may induce band-broadening distortions of the polypeptide structure.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.     

Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling

The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to −6 kJ/mol; thus not strongly favored over lipid-lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid ↔ gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few Å results in up to 10-fold increased exchange rates as compared to the ‘optimal’ match situation pointing to the regulatory role of hydrophobic coupling in lipid-protein interactions.     

Structural Polymorphism of Gramicidin A Channels: Ion Conductivity and Spectral Studies

The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded π6.3π 6.3 helix the peptide and lipid were co-dissolved in TFE prior to liposome preparation. THF and other solvents were used to form parallel (↑ ↑ π π) and antiparallel (↑ ↓ π π) double helices. Conformation of gramicidin in liposomes made by various phosphatidylcholines was monitored by CD spectroscopy, and computer analysis of the spectra obtained was performed. After fusion of gramicidin containing liposomes with planar bilayer membranes from asolectin, the histograms of single-channel conductance were obtained. The histograms had one or three distinct peaks depending on the liposome preparation. Assignment of the structure of the channel to conductance levels was made by correlation of CD data with conductance histograms. The channel-forming analogue, des(Trp-Leu)₂-gramicidin A, has been studied by the same protocol. The channel conductances of gramicidin A and the shortened analogue increase in the following order: ↑ ↓ π π 2 ↑ ↑ π π < π 6.3π6.3. Single-channels formed by double helices have higher dispersity of conductance than the π6.3π6.3 helical channel. Lifetimes of the double helical and the π6.3π6.3 helical channels are very close to each other. The data obtained were compared with theoretically predicted properties of double helices [1].     

Characterization of micellar-packaged gramicidin A channels.

The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (¹³C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (²³Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. ¹³C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T₁) demonstrated sodium interaction to be dependent on heat incorporation. The T₁ identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

Formation of ion channels by a negatively charged analog of gramicidin a

Summary O-pyromellitylgramicidin is a derivative of gramicidin in which three carboxyl groups are introduced at the terminal hydroxyl end of the peptide. Experiments with artificial lipid membranes indicate that this negatively charged analog forms ion-permeable channels in a way similar to that of gramicidin. If O-pyromellitylgramicidin is added to only one aqueous solution, the membrane conductance remains small, but increases by several orders of magnitude if the same amount is also added to the other side. In accordance with the dimer model of the channel, the membrane conductance under symmetrical conditions is proportional to the square of the aqueous concentration of O-pyromellitylgramicidin over a wide range. The ratio _PG/ _G of the single-channel conductance of O-pyromellitylgramicidin to that of gramicidin is close to unity at high ionic strength, but increases more than fivefold at smaller ionic strength (0.01m). This observation is explained in terms of an electrostatic effect of the fixed negative charges localized near the mouth of the channel. In a mixture of O-pyromellitylgramicidin and gramicidin, unit conductance steps of intermediate size are observed in addition to the conductance steps corresponding to the pure compounds, indicating the formation of hybrid channels. Hybrid channels with preferred orientation may be formed if small amounts of gramicidin and O-pyromellitylgramicidin are added to opposite sides of the membrane. These hybrid channels show a distinct asymmetry in the current-voltage characteristic.     

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.