Synthesis, characterization, and black lipid membrane studies of [7-L-alanine] gramicidin A

With a view to study the relevance of side-chain orientation in the transport of cations through a gramicidin transmembrane channel and to identify an analogue with favorable characteristics, [L-Ala7] gramicidin A was synthesized, purified, verified, and characterized by high-performance liquid chromatography, by carbon-13 and proton magnetic resonance spectra, and by circular dichroism spectra in methanol. Complete incorporation as the channel state was achieved when packaged in lysolecithin-containing lipid bilayers. The single-channel conductance data in diphytanoyllecithin/n-decane membranes are presented along with those of synthetic gramicidin A (GA). [L-Ala7] GA exhibits the highest most probable single-channel conductance so far reported for an analogue occurring at 28 pS as compared to 21 pS for GA under similar conditions. Also, a dramatic reduction in the dispersity of conducting states is observed with about 76% of the events falling in a narrow 1.75-pS conductance window as compared to about 31% of the events for GA under identical conditions. Thus, with the above characteristics, [L-Ala7]GA appears to be a very good candidate for a thorough study of ionic mechanism. The present results indicate that elements intrinsic to the channel proper are rate-limiting for GA and that there is no interfacial polarization or diffusion-controlled association at 1 M KCl and a 100-mV applied potential.     

Single-channel parameters of gramicidin a,b, and c

The single-channel conductance Λ and the mean channel lifetime τ1 of natural and synthetic gramicidins A, B, and C has been studied. Significant differences in Λ were found between gramicidin A and B; both gramicidins differ only in one amino acid (tryptophan replaced by phenylalinine). The distribution of Λ is narrow in glycerylmonooleate membranes but considerably broader in dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine membranes. The ratio of the single-channel conductances in glycerylmonooleate and dioleoyl phosphatidylcholine membranes is only about two and is considerable smaller than the conductance ratio of nonactin-mediated cation transport. This finding suggests that dipolar potentials at the membrane/solution interface have little influence on the conductance of the gramicidin channel.     

Single-channel parameters of gramicidin A,B, and C.

The single-channel conductance lambda and the mean channel lifetime gamma of natural and synthetic gramicidins A, B, and C has been studied. Significant differences in delta were found between gramicidin A and B; both gramicidins differ only in one amino acid (tryptophan replaced by phenylaline). The distribution of lambda is narrow in glycerylmonooleate membranes but considerably broader in dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine membranes. The ratio of the single-channel conductances in glycerylmonooleate and dioleoyl phosphatidylcholine membranes is only about two and is considerable smaller than the conductance ratio of nonactin-mediated cation transport. This finding suggests that dipolar potentials at the membrane/solution interface have little influence on the conductance of the gramicidin channel.     

Influence of acylation on the channel characteristics of gramicidin A.

The influence of acylation on the conductance, average duration, and channel-forming potency of channels formed by gramicidin A analogues was investigated using single-channel and multichannel techniques. Lauroyl-, myristoyl-, palmitoyl-, stearoyl-, and oleoylgramicidin A were prepared by covalent coupling of that fatty acid to the C-terminal ethanolamine group. Acylation of gramicidin A does not affect the single-channel conductance or the minichannel frequency in diphytanoylphosphatidylcholine/n-decane black lipid membranes. However, the average duration of all acylgramicidin channels was increased approximately 5-fold as compared to unmodified gramicidin A, which has a duration of 0.9 s at 200-mV applied potential. Somewhat surprisingly the rate of channel formation of the acylgramicidins is decreased relative to gramicidin A: lauroyl- and stearoylgramicidin are approximately 200 times less effective in channel formation as compared to gramicidin A. We conclude that channels formed by the acylgramicidins and by gramicidin A are structurally and conformationally equivalent.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

The structure,cation binding,transport, and conductance of Gly15-gramicidin A incorporated into SDS micelles and PC/PG vesicles

To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).     

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.     

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].     

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.     

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.     

Difference in association of Tl(I) with gramicidin A and gramicidin B in trifluoroethanol determined by Tl-205 NMR

The thallium-205 chemical shift was determined as a function of temperature for the thallium(I) complexes of gramicidin A and gramicidin B in 2,2,2-trifluoroethanol. From the difference in magnitude of the induced chemical shift it was determined that gramicidin B does not bind the Tl(I) ion as well as does gramicidin A. This result may explain the lower single-channel conductance of gramicidin B relative to gramicidin A. Cabon-13 NMR studies strongly indicate that the binding site for gramicidin A and B is at teh tryptophan end of the molecule and that replacement of tryptophan residue at position 11 in gramicidin A with a phenylalanine to form gramicidin B produces a significant structural change at the tryptophan end of the molecule, but has little effect on the N-terminus.     

Tandem gramicidin channels cross-linked by streptavidin

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.     

Synthesis and channel properties of [Tau 16]gramicidin A

Des(ethanolamine)-taurine16-gramicidin A ([Tau 16]gramicidin A) was synthesized by the solid phase method and its channel-forming behavior in planar lipid bilayers was examined. The purified monovalent anionic peptide formed channels when applied to the aqueous compartments on both sides of the bilayer, but not when applied to one side only. The single-channel conductance was measured for KCl concentrations between 0.1 and 1.0 M and was found to be higher than that of gramicidin A in each case. Single-channel lifetimes were similar to those of gramicidin A suggesting that the channels have the beta 6.3 helix structure.     

Fluorescent gramicidin derivatives for single-molecule fluorescence and ion channel measurements.

Single-molecule spectroscopies in combination with single-channel patch-clamp measurements have the potential for providing new information on ion channel gating processes. Fluorescent gramicidin derivatives could provide a means for calibrating such experiments since the structure of the open channel is known and the mechanism of gating (peptide dimerization) is generally agreed. We describe here the synthesis and characterization of two pairs of gramicidin derivatives that should prove useful for such studies. They contain robust fluorophores, undergo resonance energy transfer (FRET) when they dimerize, and have single-channel properties close to those of the wild-type channel.     

Induction of conductance heterogeneity in gramicidin channels

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.     

Ionophoric activity of truncated gramicidin A analogue in phospholipid bilayers and mitochondrial and erythrocyte membranes

Ionophoric activities of an N-terminus truncated gramicidin A (gA) analogue (mini-gramicidin) and its covalent dimer were studied in planar bilayer phospholipid membranes (BLM) using macroscopic current measurements (at high concentrations of the peptides) and single-channel recordings. As with gA-induced currents, mini-gramicidin-stimulated macroscopic currents through BLM underwent sensitized photoinactivation, i.e. were suppressed after irradiation with visible light in the presence of a photosensitizer generating singlet oxygen. The sensitivity of the tested compounds to photoinactivation descended in the following order: minigramicidin dimer > mini-gramicidin monomer > gA. The data from single-channel measurements and kinetic analysis of flash-induced photoinactivation obtained at different levels of macroscopic currents suggest that mini-gramicidin and its covalent dimer induce a variety of conducting states; their ratio depends on membrane thickness. Analysis of natural (mitochondrial and erythrocyte) membranes established that ionophoric activities of mini-gramicidin and its covalent dimer depend essentially on the membrane type.     

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

The effects of bilayer thickness and tension on gramicidin single-channel lifetime

Measurements have been made of gramicidin single-channel lifetimes in monoacylglycerol bilayers chosen so that their thickness ranged from above to below the length of the gramicidin channel. Contact angles, electrical capacities and bulk-phase interfacial tensions have also been determined for these systems. The mean channel lifetime decreased with the hydrocarbon thickness of the membrane until the latter reached 2.2 nm, after which the lifetime was relatively constant. A theoretical model has been proposed which relates the mean channel lifetime (or dissociation constant) to both the thickness and the tension of the bilayers. The analysis of the present results and of those of previous studies has led to the idea that aggregates of water molecules may play an important r?le in the dissociation of the gramicidin channel.     

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.     

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.