Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.     

Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

Water permeability of gramicidin A-treated lipid bilayer membranes

In membranes containing aqueous pores (channels), the osmotic water permeability coefficient, P f, is greater than the diffusive water permeability coefficient, P d. In fact, the magnitude of P f/P d is commonly used to determine pore radius. Although, for membranes studied to date, P f/P d monotonically declines with decreasing pore radius, there is controversy over the value it theoretically assumes when that radius is so small that water molecules cannot overtake one another within the channel (single-file transport). In one view it should equal 1, and in another view it should equal N, the number of water molecules in the pore. Gramicidin A forms, in lipid bilayer membranes, narrow aqueous channels through which single-file transport may occur. For these channels we find that P f/P d approximately 5. In contrast, for the wider nystatin and amphotericin B pores, P f/P d approximately 3. These findings offer experimental support for the view that P f/P d = N for single-file transport, and we therefore conclude that there are approximately five water molecules in a gramicidin A channel. A similar conclusion was reached independently from streaming potential data. Using single-channel conductance data, we calculate the water permeability of an individual gramicidin A channel. In the Appendix we report that there is a wide range of channel sizes and lifetimes in cholesterol-containing membranes.     

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.     

Brief closures of gramicidin A channels in lipid bilayer membranes

Brief closures, so called flickers, gramicidin A channels were observed for glycerol monooleate/n-decane membranes for cesium chloride and hydrochloric acid solutions. The flickers, similar in nature to the flickers observed for physiological channels, were of the order of 1 ms and the interval between flickers was of the order of 50 ms. The flicker-duration and interval between flickers both decrease with voltage. The field dependence of the flickers is consistent with the hypothesis that the membrane forms a dimple when accomodating a dimer in the membrane and that the monomers, on breaking up, are associated over displacements of the order of 2 nm. For similar measurements for glycerol monoleate/hexadecane membranes only rare occurrences of flickers were observed. It is suggested that the flicker phenomenon is governed by the physical and chemical properties of the membrane and the influence of membrane thickness and interfacial free energy is emphasized.     

Channel formation kinetics of gramicidin A in lipid bilayer membranes

Summary Previous studies have given evidence that the active form of gramicidin A in lipid bilayer membranes is a dimer which acts as an ion channel; it has been further shown that the mean lifetime of the channel strongly depends on the membrane thickness. As the thickness slightly decreases when a voltage is applied to the membrane, the equilibrium between conducting dimers and nonconducting monomers may be displaced by a voltage jump. From the relaxation of the electrical current after the voltage jump, information about the kinetics of channel formation is obtained. For a dioleoyllecithin/n-decane membrane the rate constant of association is found to be 2×10¹⁴ cm² mole^–1 sec^–1, which is by three orders of magnitude below the limiting value of a diffusion-controlled reaction in a two-dimensional system. The dissociation rate constant is equal to 2 sec^–1, a value which is consistent with the channel lifetime as obtained from electrical fluctuation measurements.     

Effect of bilayer lipid membrane thickness, composition, and tension on gramicidin channel parameters

Dependence of channel parameters formed by gramicidin A (conductivity and mean life time) on thickness, composition and tension of planar bilayer lipid membranes (BLM) was studied. BLM were obtained from solutions of alpha-monoglycerides of fatty acids in n-alkanes. It has been shown that channel conductivity depends on the length of lipid radical hydrocarbon and is insensitive to the isomerization of lipid and to the change of solvent. There was no direct relationship between the life time, thickness and composition of BLM. Logarithm tau for all the systems studied is proportional to BLM tension, which points to a significant role of surface phenomena in the formation by grammicidine A of a conducting pore in the lipid bilayer.     

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.     

Stimulation of gramicidin incorporation into lipid bilayer membranes by ultrasound

Ultrasound effect on gramicidin incorporation into a bilayer lipid membrane has been investigated. The observed increase in the channel opening frequency points to the incorporation rate growth due to the thickness diminishing of near-membrane non-stirred layers. The dependence of ultrasound intensity on the layer thickness is presented.     

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.     

Two phases of gramicidin photoinactivation in bilayer lipid membranes in the presence of a photosensitizer

The kinetics of the light-induced decrease in the gramicidin-mediated current across a bilayer lipid membrane in the presence of a photosensitizer has been shown to include a slow phase with a characteristic time of the order of 1 s and a fast phase. Based on the dependence of the slow phase relative amplitude and characteristic time on the gramicidin-mediated stationary conductance we concluded that the slow phase reflected the establishment of an equilibrium between gramicidin monomers and dimers in the membrane after the distortion of this equilibrium resulting from modification of a portion of gramicidin molecules by reactive oxygen species generated upon excitation of the photosensitizer. The dependence of the fast phase contribution to the overall kinetics on the stationary conductance allowed us to conclude that the fast phase is associated with transition of gramicidin dimers into a nonconducting state. The characteristic time of the fast phase measured with nanosecond laser excited pulses is 1.5 ms. The slow phase of the decrease in the gramicidin-mediated current was considerably decelerated in the presence of Rose Bengal. The results obtained indicate that adsorption of Rose Bengal on the bilayer interface leads to a reduction of the dipole potential drop at the membrane-solution boundary, similarly to the action of phloretin.     

Interpretation of fluctuation spectra in lipid bilayer simulations

Atomic resolution and coarse-grained simulations of dimyristoylphosphatidylcholine lipid bilayers were analyzed for fluctuations perpendicular to the bilayer using a completely Fourier-based method. We find that the fluctuation spectrum of motions perpendicular to the bilayer can be decomposed into just two parts: 1), a pure undulation spectrum proportional to q⁻⁴ that dominates in the small-q regime; and 2), a molecular density structure factor contribution that dominates in the large-q regime. There is no need for a term proportional to q⁻² that has been postulated for protrusion fluctuations and that appeared to have been necessary to fit the spectrum for intermediate q. We suggest that earlier reports of such a term were due to the artifact of binning and smoothing in real space before obtaining the Fourier spectrum. The observability of an intermediate protrusion regime from the fluctuation spectrum is discussed based on measured and calculated material constants.     

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.     

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.