Enzyme-bound formylvaline and formylvalylglycine; an initiation complex for gramicidin A biosynthesis.

Gramicidin A is an antibiotic peptide produced by Bacillus brevis ATCC 8185, which also produces tyrocidines. An attempt was made to establish a cell-free enzyme system for gramicidin A synthesis. An enzyme fraction, Component I, was partially purified from crude extracts of the organism and proven to be involved in the synthesis of the formyl-Val-Gly- region of gramicidin A. The initiation of gramicidin A biosynthesis is a function of Component I, which activates valine and binds it as a thioester, and further formylates it in the presence of formyltetrahydrofolic acid. The formylvaline thus synthesized is transferred to the glycine moiety, which is also thioesterified to Component I. Elongation of the peptide chain takes place by a mechanism similar to those found for tyrocidines, gramicidin S, and bacitracin.     

Conformation of gramicidin a in water: inference from analysis of hydrogen/deuterium exchange behavior by matrix assisted laser desorption ionization mass spectrometry

Gramicidin A (the major component of gramicidin D) is a highly hydrophobic peptide with very little solubility in water. Hence, the conformation of this peptide has been extensively investigated in organic solvents and model membranes, but not in water. The peptide adopts a beta6.3-helical conformation in the monomeric and dimeric forms. We have investigated the conformation of gramicidin A in water by monitoring hydrogen-deuterium exchange by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Our results indicate that gramicidin A is monomeric and exists in a highly folded conformation. The metal ion bound forms are clearly discernible in the monomers. The presence of the dimeric form is not observed. It is unlikely this is due to the operating conditions or the method used, as both hetero- and homodimers in gramicidin D are detected when methanol is used as a solvent. The present study also establishes that the linear gramicidins retain a history of solvent environment when ions are generated by matrix-assisted laser desorption ionization and analyzed by time-of-flight.     

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.     

Gramicidin K, a new linear channel-forming gramicidin from Bacillus brevis

A new gramicidin has been isolated from a commercial mixture of gramicidins A, B, and C. This new molecule, designated gramicidin K, contains formyl and ethanolamine blocking groups, has a molecular weight approximately 20% higher than gramicidin A, and is strongly retained on reversed-phase liquid chromatographic columns. Gramicidin K can be resolved into two components, one of which contains tyrosine. In lipid bilayer membranes, both components form channels of considerably longer lifetime and somewhat lower conductance than gramicidin A. Gramicidin K appears to be a lipopeptide that consists of a fatty acyl chain attached to the ethanolamine of gramicidin A.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Protein stability and conformational rearrangements in lipid bilayers: linear gramicidin, a model system.

The replacement of four tryptophans in gramicidin A by four phenylalanines (gramicidin M) causes no change in the molecular fold of this dimeric peptide in a low dielectric isotropic organic solvent, but the molecular folds are dramatically different in a lipid bilayer environment. The indoles of gramicidin A interact with the anisotropic bilayer environment to induce a change in the molecular fold. The double-helical fold of gramicidin M, as opposed to the single-stranded structure of gramicidin A, is not compatible with ion conductance. Gramicidin A/gramicidin M hybrid structures have also been prepared, and like gramicidin M homodimers, these dimeric hybrids appear to have a double-helical fold, suggesting that a couple of indoles are being buried in the bilayer interstices. To achieve this equilibrium structure (i.e., minimum energy conformation), incubation at 68 degrees C for 2 days is required. Kinetically trapped metastable structures may be more common in lipid bilayers than in an aqueous isotropic environment. Structural characterizations in the bilayers were achieved with solid-state NMR-derived orientational constraints from uniformly aligned lipid bilayer samples, and characterizations in organic solvents were accomplished by solution NMR.     

Effect of gramicidin A on the dipole potential of phospholipid membranes.

The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential.     

Stability of an ion channel in lipid bilayers: implicit solvent model calculations with gramicidin

Gramicidin is a helical peptide, 15 residues in length, which dimerizes to form ion-conducting channels in lipid bilayers. Here we report calculations of its free energy of transfer from the aqueous phase into bilayers of different widths. The electrostatic and nonpolar contributions to the desolvation free energy were calculated using implicit solvent models, in which gramicidin was described in atomic detail and the hydrocarbon region of the membrane was described as a slab of hydrophobic medium embedded in water. The free energy penalties from the lipid perturbation and membrane deformation effects, and the entropy loss associated with gramicidin immobilization in the bilayer, were estimated from a statistical thermodynamic model of the bilayer. The calculations were carried out using two classes of experimentally observed conformations: a head-to-head dimer of two single-stranded (SS) beta-helices and a double-stranded (DS) intertwined double helix. The calculations showed that gramicidin is likely to partition into the bilayer in all of these conformations. However, the SS conformation was found to be significantly more stable than the DS in the bilayer, in agreement with most of the experimental data. We tested numerous transmembrane and surface orientations of gramicidin in bilayers of various widths. Our calculations indicate that the most favorable orientation is transmembrane, which is indeed to be expected from a channel-forming peptide. The calculations demonstrate that gramicidin insertion into the membrane is likely to involve a significant deformation of the bilayer to match the hydrophobic width of the peptide (22 A), again in good agreement with experimental data. Interestingly, deformation of the bilayer was induced by all of the gramicidin conformations.     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

Photolysis of gramicidin A channels in lipid bilayers

We have tested the hypothesis that peptide tryptophan groups can control the ionic conductance of transmembrane channels. We report here that single gramicidin A channels change conductance state when the peptide tryptophans are flash photolyzed with ultraviolet light. The current flow through planar lipid bilayers containing multiple gramicidin A channels decreases irreversibly when exposed to ultraviolet light. The current-loss action spectrum peaks sharply at the 280 nm absorption maximum of the gramicidin A tryptophans. Gramicidin channel sensitivity to ultraviolet light is found to be about 20-fold higher than that of frog node sodium channels which is even more than expected based on the high tryptophan content of gramicidin. Channels which survive an ultraviolet light exposure exist in a wide variety of different low-conductance forms. The broad distribution of the single channel conductance of these partially photolyzed channels is attributable to the loss of different combinations of the dimer's normal complement of eight tryptophans per channel. Flash photolysis of single channels results in discrete conductance state changes. Partially photolyzed single channels manifest a further conductance cascade when exposed to a second flash of ultraviolet light. Analysis of the photolysis conductance turn-off process indicates that gramicidin A is a multistate electrochemical unit where the peptide tryptophan groups can modulate the flow of ions through the transmembrane channel.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

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.     

The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS⁴ gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes.     

Formation of ionic channels in black lipid membranes by succinic derivatives of Gramicidin A

Summary Different succinyl derivatives of Gramicidin A were synthesized and their activity was investigated with different methods on lipid bilayer membranes. The succinyl derivatives of Gramicidin A can be classified as three different types, the O-succinyl derivative, the N-succinyl derivative and the N-O-succinyl derivative of Gramicidin A. An O-pyromellityl-N-succinyl gramicidin was synthesized which can be attributed to the latter class. It was found that O-succinyl gramicidin behaves like the unmodified Gramicidin A despite a charge effect on single-channel conductance, arising from the negative charge of the succinic residue, at the mouth of the channel. The activity of N-succinyl and N-O-succinyl gramicidin and of O-pyromellityl-N-succinyl-gramicidin depends strongly on the pH of the electrolyte solution. It is demonstrated that at low pH (5) the N-succinyl derivatives show high activity, whereas at high pH (7) the activity is sharply reduced or disappears totally. From these experiments it can be concluded that, for the formation of a dimeric gramicidin channel, the hydrogen of the formyl group can be replaced by a protonated carboxylic group of a succinic residue.Further results, obtained by measurement of the single-channel conductance and of the reaction rate constants for the channel formation, are discussed in terms of the structural basis of the single stranded model for the gramicidin channel. On this basis the double stranded helix can be, excluded and an interesting head-to-head single stranded (_L,D) helical channel is described which contains carboxyl groups at the head-to-head junction.     

Supramolecular organization of lysophosphatidylcholine-packaged Gramicidin A

Heat derived gramicidin A'/L-alpha-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A' to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A' molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50-60 A periodicity along the row and with 70-80 A distance between rows. An idealized working model for the incorporated state is presented.     

Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Gramicidin A directly inhibits mammalian Na+/K+-ATPase

The linear pentadecapeptide gramicidin A forms an ion channel in the lipid bilayer to selectively transport monovalent cations. Nevertheless, we have surprisingly found that gramicidin A directly inhibits mammalian Na(+)/K(+)-ATPase. Gramicidin A inhibited ATP hydrolysis by Na(+)/K(+)-ATPase from porcine cerebral cortex at the IC(50) value of 8.1 microM, while gramicidin S was approximately fivefold less active. The synthetic gramicidin A analog lacking N-terminal formylation and C-terminal ethanolamine exhibited a weaker inhibitory effect on the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase than gramicidin A, indicating that these end modifications are necessary for gramicidin A to inhibit Na(+)/K(+)-ATPase activity. Moreover, Lineweaver-Burk analysis showed that gramicidin A exhibits a mixed type of inhibition. In addition to the most well-studied ionophore activity, our present study has disclosed a novel biological function of gramicidin A as a direct inhibitor of mammalian Na(+)/K(+)-ATPase activity.