Derivatives Of Gramicidin Research Articles

Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes

Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA<[Lys5]gA<[Lys1]gA<[Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation.

Electrochemical impedance spectroscopy in label-free biosensor applications: multivariate data analysis for an objective interpretation

Electrochemical impedance spectroscopy plays an important role in biosensor science thanks to the possibility of finding specific information from processes with different kinetics at a chosen electrode potential in one experiment. In this paper we briefly discuss label-free impedimetric biosensors described in the literature. A novel method for neutral interpretation of impedance data is presented that includes complex number chemometrics. Three examples are given based on impedance measurements on synthetic biomembranes, in this case a lipid monolayer deposited on a mercury electrode. The interaction of various compounds with the monomolecular lipid layer is illustrated with the following: (1) different concentrations of magainin (Geladi et al. in Proc. Int. Fed. Med. Biomed. Eng. 9:219-220, 2005); (2) different derivatives of gramicidin A (Lindholm-Sethson et al. in Langmuir 24:5029-5032, 2007), and (3) an antimicrobial peptide (Ringstad et al. in Langmuir 24:208-216, 2008).

Ring‐Extended Derivatives of Gramicidin S with Furanoid Sugar Amino Acids in the Turn Region Have Enhanced Antimicrobial Activity

A series of ring-extended gramicidin S (GS) derivatives containing furanoid sugar amino acids were evaluated. Although the extended GS derivatives have a less well-defined secondary structure as determined by NMR and CD, some derivatives show an improved biological profile, namely, an increased antibacterial activity and decreased hemolytic activity.

Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

Ion channel-forming peptides and proteins offer tremendous opportunities for fundamental and applied studies of function on individual molecules. An ongoing challenge in ion channel research is the lack of simple and accessible synthetic methods to engineer pores with tailored chemical and physical properties. This paper describes a practical synthetic route to rapidly generate C-terminally modified derivatives of gramicidin A (gA), an ion channel-forming peptide, through the use of two chemically reactive gA-based building blocks. These amine- and azide-containing building blocks can react readily with typical substrates for amidation and 1,3-dipolar cycloaddition ("click") reactions to present molecules with desired structure and functionality near the opening of a gA pore. These derivatives of gA are stable under typical aqueous conditions for ion channel recordings and retain characteristic single ion channel conductance properties in planar lipid bilayers. Additionally, the synthetic methods described here afford useful quantities of these gA derivatives in good purity and yield with minimal purification. We demonstrate that derivatives of gA can be used for studying, in situ, a change in conductance through a channel upon performing a "click" reaction on an azide moiety attached to the gA pore. We also demonstrate that these gA-based building blocks can be used to construct sensors for the recognition of specific protein-ligand binding interactions in solution. This widely accessible, enabling synthetic methodology represents a powerful new tool to study relationships between chemical structure and function on the single molecule level.

Designing Nanosensors Based on Charged Derivatives of Gramicidin A

Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We recently reported the possibility to detect chemical modifications on individual molecules by monitoring a change in the single ion channel conductance of derivatives of gramicidin A (gA) upon reaction with analytes in solution. These peptide-based nanosensors detect reaction-induced changes in the charge of gA derivatives that were engineered to carry specific functional groups near their C-terminus.1 Here, we discuss five key design parameters to optimize the performance of such chemomodulated ion channel sensors. In order to realize an effective sensor that measures changes in charge of groups attached to the C-terminus of a gA pore, the following conditions should be fulfilled: (1) the change in charge should occur as close to the entrance of the pore as possible; (2) the charge before and after reaction should be well-defined within the operational pH range; (3) the ionic strength of the recording buffer should be as low as possible while maintaining a detectable flow of ions through the pore; (4) the applied transmembrane voltage should be as high as possible while maintaining a stable membrane; (5) the lipids in the supporting membrane should either be zwitterionic or charged differently than the derivative of gA. We show that under the condition of high applied transmembrane potential (>100 mV) and low ionic strength of the recording buffer (< or =0.10 M), a change in charge at the entrance of the pore is the dominant requirement to distinguish between two differently charged derivatives of gA; the conductance of the heterodimeric gA pore reported here does not depend on a difference in charge at the exit of the pore. We provide a simple explanation for this asymmetric characteristic based on charge-induced local changes in the concentration of cations near the lipid bilayer membrane. Charge-based ion channel sensors offer tremendous potential for ultrasensitive functional detection since a single chemical modification of each individual sensing element can lead to readily detectable changes in channel conductance.

Multivariate Data Analysis for Enhanced Interpretation of Electrochemical Impedance Spectra of Gramicidin−Ion Interactions in Phospholipid Monolayers

This article describes a multifrequency electrochemical impedance study of phospholipid monolayers on a mercury drop electrode in solutions containing electrolytes and gramicidin derivatives: gramicidin A (gA), gramicidin-BOC (g-BOC), and desformylgramicidin (g-des). The impedance spectra have been studied individually (univariate approach) and also transformed using a multivariate data reduction method (multivariate approach). It was shown that the two approaches are complementary. Thus the formation of K+-conducting channels is observed in gA only, and these channels can be distinguished from an interaction of all gramicidin derivatives with Mg2+. An unknown peptide interaction in the monolayer was observed on a slow time scale.

Interaction of gramicidin derivatives with phospholipid monolayers.

A study of the interaction of gramicidin A (gA), tert-butyloxycarbonyl-gramicidin (g-BOC), and desformyl gramicidin (g-des) with dioleoyl phosphatidylcholine (DOPC) and DOPC/phosphatidylserine (PS) mixed monolayers on a mercury electrode is reported in this paper. Experiments were carried out in electrolytes KCl (0.1 mol dm(-3)) and Mg(NO3)2 (0.05 mol dm(-3)). The channel-forming properties of the gramicidins were studied by following the reduction of Tl(I) to Tl(Hg). The frequency dependence of the complex impedance of coated electrode surfaces in the presence and absence of the gramicidins was estimated between 65,000 and 0.1 Hz at potentials of -0.4 V versus Ag/AgCl with 3.5 mol dm(-3) KCl. Epifluorescence microscopy was used to qualitatively correlate the interaction of the gramicidin peptides with dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) at the air-water interface. gA was shown to form Tl+ conducting channels in a DOPC monolayer, while g-BOC and g-des did not. In DOPC-30% PS (DOPC-0.3PS) layers, there is a marked increase in channel activity of all three gramicidin derivatives. None of the peptides facilitate the permeability of the DOPC-0.3PS layer to Cd2+. All three peptides interact with the layer as shown by capacitance-potential curves and impedance spectroscopy indicated by penetration of the peptide into the dielectric, an increase in surface "roughness", and an increased significance of low-frequency relaxations. The order of interaction is gA > g-des > g-BOC. The epifluorescence study of DPPC and DPPG layers at the air-water interface shows a selective action of the different gramicidins.

Simultaneous Optical and Electrical Recording of Single Gramicidin Channels

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 × 10 −8 cm 2s −1. Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 × 10 −8 cm 2s −1. These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.

Stereochemistry of protected ornithine side chains of gramicidin S derivatives: X-ray crystal structure of the bis-Boc-tetra-N-methyl derivative of gramicidin S.

An X-ray crystallographic analysis of the bis-Ndelta-Boc-tetra-Nalpha-methyl derivative of gramicidin S, cyclo(-Val-MeOrn(Boc)-Leu-d-MePhe-Pro-)2, was undertaken successfully (R-factor = 0.088). As expected, the main chain adopts an antiparallel pleated beta-sheet conformation, but the pleated sheet is slightly twisted, and the sense of twisting is opposite to that found in the reported crystal structures of the gramicidin S-urea complex and the bis-Ndelta-(trichloroacetyl) and bis-Ndelta-(m-bromobenzoyl) derivatives of gramicidin S. In agreement with the observed resistance toward N-methylation, the urethane NH groups of the protected Orn side chains are hydrogen bonded to the carbonyl groups of the d-Phe residues. However, the side-chain-main-chain hydrogen bonding is in the i --> i - 3 mode, although hydrogen bonding in the i --> i + 2 mode was deduced from a 1H NMR study of protected gramicidin S derivatives and was actually found in the crystal structures of the diacylated gramicidin S.

Gramicidin derivatives as membrane-based pH sensors

Ion channels provide a means for sensitive pH measurement at membrane interfaces. Detailed knowledge of the structure and function of gramicidin channels permits the engineering of pH-sensitive derivatives. Two derivatives, gramicidin-ethylenediamine and gramicidin-histamine, are shown to exhibit pH-dependent single-channel behaviour over the pH ranges 9–11 and 6.5–8.5, respectively. Thermal isomerization of a carbamate group at the entrance of the channels leads to a pattern of steps in single-channel recordings. The size of the steps depends on the time-averaged degree of protonation of the appended group (ethylenediamine or histamine). Measurement of the size of the steps thus permits single-molecule pH sensing under symmetrical pH conditions or in the presence of a pH gradient.

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.

Preparation and metal ion-binding properties of gramicidin S derivatives carrying picolinoyl groups on the ornithine side chains

Derivatives of gramicidin S (GS) in which one or both of the two ornithine side chains were picolinoylated were prepared. The dipicolinoyl derivative [Orn(PyCO)2,2′]GS 1 formed a 1∶1 complex with Cu2+ and Zn2+ in MeOH, while in the case of the monopicolinoyl derivative [Orn(Boc)2,Orn(PyCO)2′]GS (2) stepwise formation of 1∶1 and 2∶1 (2∶Cu2+) complexes was observed. The formation constant of the Cu2+-mediated dimeric species of compound 2 was larger than those of the corresponding linear compounds possessing the partial structure of macrocycle 2. The corresponding tetra-N-methyl derivative [MeOrn(Boc)2,MeOrn(PyCO)2′,D-MePhe4,4′]GS 3 also showed lower stability of the 2∶1 complex compared with compound 2, which suggested the presence of a β-sheet-type intermolecular H-bonding interaction between the two molecules of macrocycle 2 in the 2∶1 complex.

Stereochemistry of protected ornithine side chains in gramicidin S derivatives and their resistance to N-methylation

Derivatives of gramicidin S (GS) and its mono- and di-d-cyclohexylalanined-Cha) analogs possessing various protecting groups on Orn side chains were prepared.1H NMR spectra of the unsymmetrically protected analogs [Orn(X)2, Orn(X′)2′,d-Cha4]GS were similar to the composites of the spectra of the symmetrical derivatives [Orn(X)2,2′,d-Cha4,4′]GS and [Orn(X′)2,2′]Gs, revealing the proximity of the protecting groups of NδH of Orn residues at the 2 and 2′ positions to the side chains ofd-Phe (ord-Cha) residues at the 4 and 4′ positions, respectively. The results indicated the presence of H-bonds between the N°H of Orn and the carbonyl ofd-Phe residues in the i→i+2 sense and not in i→i-3, which was also supported by the ROESY analysis. The substantially strong H-bonds can explain the observed resistance of the urethane NH of the Orn side chains in the GS derivatives to the N-methylation with CH3I−Ag2O in DMF.

C-terminal amino groups facilitate membrane incorporation of gramicidin derivatives

Gramicidin derivatives with (positively-charged) C-terminal amino groups are found to incorporate readily and refold quickly when added to dioleoylphosphatidylcholine lipid vesicles from concentrated methanol solutions. Neutral and negatively-charged derivatives do not.

Effect of salt and membrane fluidity on fluorophore motions of a gramicidin C derivative

We have used fluorescence spectroscopy to investigate the effect of salt and membrane fluidity on the rotational motion of a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) derivative of gramicidin C (dansyl-gC) in dimyristoylphosphatidylcholine bilayers, under conditions where the peptide is a formyl-NH to formyl-NH terminal dimer, and in octyl glucoside micelles, where the peptide is an intertwined helical dimer. Energy-transfer experiments showed no changes in either conformation or dimer aggregation under the conditions explored here (15-40 degrees C, 1-350 bar, 0-0.33 M Mg2+, and 0-1 M Na+). The addition of permeable (Na+) or nonpermeable (Mg2+) ions did not affect the temperature or pressure behavior of dansyl rotation. However, fluorescence lifetime measurements indicated an increase in solvent accessibility in the presence of sodium. In bilayers, the temperature dependence of the fluorescence polarization and lifetime shows strong interactions between the dansyl residue and the peptide, and at no time did the dansyl motions become solvent controlled as has been observed for aqueous solvent peptides [Scarlata, S. F., Rholam, M., & Weber, G. (1984) Biochemistry 23, 6789]. In micelles, the change in rotational motion with temperature followed solvent expansion, showing that in this case the dansyl residue does not associate extensively with the peptide. Our results indicate that because of the extensive coupling between the dansyl residue and the rest of the peptide, membrane fluidity does not play a major role in controlling side-chain motions.

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

Mechanism of the uncoupling of oxidative phosphorylation by gramicidin

The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of delta mu H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and delta pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

ChemInform Abstract: Synthesis of a Heavy‐Atom Derivative of Gramicidin S (GS), (D‐Phe(4‐Br)4,4′)‐GS, by a Novel Method.

AbstractThe crystalline title compound (II) can be synthesized by cyclization of the linear peptide (I) which in turn is prepared by the solid‐phase method starting from the protected Pro unit.

The Synthesis of a Heavy-Atom Derivative of Gramicidin S (GS), [d-Phe(4-Br)4,4′]-GS, by a Novel Method

Abstract A new approach for the preparation of cyclic peptides was successfully applied to the synthesis of cyclo(–Val–Orn–Leu–d–Phe(4-Br)–Pro–Val–Orn–Leu–d–Phe (4-Br)–Pro–) ([d–Phe(4-Br)4,4′]-GS). [d–Phe(4-Br)4,4′]–GS showing antibacterial activity was obtained as crystals.

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608–619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(- X- d-Leu- d-Lys- d-Leu- l-Pro -) 2 , where X = Gly, d-Leu and d-cyclohexylalanine ( d -c HxAla ); (2) N, N′-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a β-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = d -c HxAla induced the efflux of K + through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K + from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.