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.     

Supramolecular organization of lysophosphatidylcholine-packaged gramicidin A′

Heat derived gramicidin A′/l-α-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 Å periodicity along the row and with 70–80 Å distance between rows. An idealized working model for the incorporated state is presented.     

Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes.

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin. The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1-2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin.The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1–2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.     

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.     

Water structure in the Gramicidin A transmembrane channel

The interaction energy and the structure of water molecules either inside the Gramicidin A transmembrane channel or at its two extremities is examined with the use of iso-energy maps and Monte Carlo simulations. The shape of the channel as experienced by water is analyzed in detail. Variations in the hydration structure due to the presence of a Na+ ion placed at several positions along the channel are simulated, analyzed and discussed. Preliminary data on Li+ and K+ interacting with Gramicidin A and the system of water molecules are reported. The Gramicidin A atomic coordinates have been taken from Urry's recent papers.     

Lysolecithin and cholesterol interact stoichiometrically forming bimolecular lamellar structures in the presence of excess water, of lysolecithin or cholesterol.

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 degrees C unlike lysolecithin alone, which forms micelles, Thes results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggest another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin-cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.     

Water Structure in the Gramicidin A Transmembrane Channel

Abstract

The interaction energy and the structure of water molecules either inside the Gramicidin A transmembrane channel or at its two extremities is examined with the use of iso-energy maps and Monte Carlo simulations. The shape of the channel as experienced by water is analyzed in detail. Variations in the hydration structure due to the presence of a Na+ ion placed at several positions along the channel are simulated, analyzed and discussed. Preliminary data on Li+ and K+ interacting with Gramicidin A and the system of water molecules are reported. The Gramicidin A atomic coordinates have been taken from Urry's recent papers.     

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.     

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.     

Action of two classes of channel-forming synthetic peptides on lipid bilayers

Single channel conductances were observed in lipid bilayers by the action of three Dialkylamide derivatives of Gramicidin and two polymers of the tripeptide (Ala-Ala-Gly). The temperature and thickness dependence showed conformational changes indicating multiple states of the helical structure.     

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.     

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.     

Hydrophobic mismatch in gramicidin A'/lecithin systems.

Gramicidin A' (GA') has been added to three lipid systems of varying hydrophobic thicknesses: dimyristoyllecithin (DML), dipalmitoyllecithin (DPL), and distearoyllecithin (DSL). The similarity in length between the hydrophobic portion of GA' and the hydrocarbon chains of the lipid bilayers has been studied by using 31P and 2H NMR. Hydrophobic mismatch has been found to be most severe in the DML bilayer system and minimal in the case of DSL. In addition, the effects of hydrophobic mismatch on the cooperative properties of the bilayer have been obtained from 2H NMR relaxation measurements. The results indicate that incorporation of the peptide into the bilayer disrupts the cooperative director fluctuations characteristic of pure multilamellar lipid dispersions. Finally, the GA'/lecithin ratio at which the well-known transformation from bilayer to reverse hexagonal (HII) phase occurs (Van Echteld et al., 1982; Chupin et al., 1987) is shown to depend on the acyl chain length of the phospholipid. A rationale is proposed for this chain length dependence.     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

Water structure in the Gramicidin A transmembrane channel

The interaction energy and the structure of water molecules either inside the Gramicidin A transmembrane channel or at its two extremities is examined with the use of iso-energy maps and Monte Carlo simulations. The shape of the channel as experienced by water is analyzed in detail. Variations in the hydration structure due to the presence of a sodium ion placed at several positions along the channel are simulated, analyzed and discussed. Preliminary data on Li+ and K+ interacting with Gramicidin A and the system of water molecules are reported. The Gramicidin A atomic coordinates have been taken from Urry's recent papers (Urry, D.W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676 and Urry, D.W., Trapane, T.L. and Prasad, K.U. (1982) Int. J. Quant. Chem. Quant. Biol. Symp. 9, 31-40).     

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.     

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.