Analogs Of Gramicidin Research Articles

Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes.

Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively strong interactions with anionic phospholipids, may explain why GS14dK(4) retains relatively high antimicrobial activity. However, its low hemolytic activity is probably largely attributable to its low propensity to penetrate and/or partition into cholesterol-containing zwitterionic lipid membranes.

Exploring relationships between mimic configuration, peptide conformation and biological activity in indolizidin-2-one amino acid analogs of gramicidin S.

Indolizidin-2-one amino acids (I2aas, 6S- and 6R-1) possessing 6S- and 6R-ring-fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution-phase and solid-phase techniques were used to synthesize three analogs with I2aa residues in place of the d-Phe-Pro residues at the turn regions of GS: [(6S)-I2aa4-5,4'-5']GS (2), [Lys2,2',(6S)-I2aa4-5,4'-5']GS (3) and [(6R)-I2aa4-5,4'-5']GS (4). Although conformational analysis of [I2aa4-5,4'-5']GS analogs 2-4 indicated that both ring-fusion stereoisomers of I2aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)-I2aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)-I2aa analog 4, suggesting a greater propensity for the (6S)-diastereomer to adopt the beta-turn/antiparallel beta-pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)-I2aa analog 2 exhibited significantly better potency than the (6R)-I2aa diastereomer 4. Relative to GS, [(6S)-I2aa4-5,4'-5']GS (2) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)-I2aa diastereomer. The synthesis and evaluation of GS analogs 2-4 has furnished new insight into the importance of ring-fusion stereochemistry for turn mimicry by indolizidin-2-one amino acids as well as novel antimicrobial peptides.

Probing the structural determinants of type II' beta-turn formation in peptides and proteins.

The structural determinants of type II' beta-turns were probed through a comprehensive CD, NMR, and molecular dynamics analysis of 10 specially designed beta-hairpin peptides. The peptide model used in this study is a synthetic, water-soluble, 14-residue cyclic analogue of gramicidin S which contains two well-defined type II' beta-turns connected by a highly stable, amphipathic, antiparallel beta-sheet. A variety of coded and noncoded amino acids were systematically substituted in one of the two type II' turns to analyze the effects of backbone chirality, side-chain steric restriction, and side-chain/side-chain interactions. beta-Sheet content (as measured through a variety of experimental methods), molecular dynamics, and 3D structural analysis of the turn regions were used to assess the effects of each amino acid substitution on type II' beta-turn stabilization. Our results demonstrate that backbone heterochirality, which determines equatorial and axial side-chain orientation at the i+1 and i+2 residues of type II' turns, may account for up to 60% of type II' beta-turn stabilization. Steric restriction through side-chain N-alkylation appears to enhance type II' beta-turn propensity and may account for up to 20% of type II' beta-turn stabilization. Finally, aromatic/proline side-chain interactions appear to account for approximately 10% of type II' beta-turn stabilization. We believe this information could be particularly useful for the prediction of beta-turn propensity, the development of peptide-based drugs, and the de novo design of peptides, proteins, and peptidyl mimetics.

Molecular structure heterogeneity of gramicidin analogs incorporated into SDS micelles: a NMR study

The effects of glycine, alanine and valine substitution for leucine upon the species heterogeneity of gramicidin A incorporated into SDS micelles have been investigated. The sequential replacement of the leucine residues at positions 10, 12 and 14 produced multiple β-helical forms of the gramicidin A analogs as shown by 1H NMR spectroscopy. These results reveal the complexity of the interaction of amino acid residue side chain with the SDS micelle environment in controlling the formation of a single channel species or multiple helical forms. Clearly, a potential problem may arise if one assumes that the solubilization of the peptide, gramicidin, in micelles indicates the existence of a single form of the peptide.

Diastereoisomeric analogues of gramicidin S: structure, biologicalactivity and interaction with lipid bilayers.

Analogues of a structurally equivalent version of theantimicrobial decameric cyclic peptide gramicidin S, GS10 [cyclo-(Val-Lys-Leu-d-Tyr-Pro)(2)], were designed to study theeffect of distortion in the beta-sheet/beta-turn structure of thecyclic peptide on its biological activity. In one approach, thehydrophobic nature of GS10 was conserved, and single amino acids in itsbackbone were replaced systematically with their correspondingenantiomers to give five diastereoisomeric analogues. In a relatedapproach, a more basic and hydrophilic analogue of GS10 [cyclo-(Lys-Val-Lys-d-Tyr-Pro(5)-Lys-Leu-Lys-d-Tyr-Pro(10))], together with two of itsmonosubstituted diastereoisomeric analogues (featuring d-Lys(1) or d-Val(2) respectively), weresynthesized. CD spectra were measured in a variety of environments,i.e. aqueous, aqueous trifluoroethanol and those containing SDSmicelles or phospholipid vesicles. In comparison with GS10 spectra, CDspectra of both groups of analogues in these environments exhibitedstructural distortion. Moreover, compared with GS10, antimicrobial andhaemolytic activities of the analogues were drastically decreased, implying the existence of a threshold minimum amphipathicity foreffective biological activity. However, in both groups of analogues,there was a correlation between amphipathicity and antimicrobial andhaemolytic activities. In the second group of analogues, bothelectrostatic and hydrophobic factors were related to theirantimicrobial and haemolytic activities. In order to gain an insightinto the nature of the biological activity of the two classes of cyclicpeptides, the relationship of their structure to interaction with lipidmembranes, and the implied mechanisms, were analysed in some detail inthe present study.

Noncontact Dipole Effects on Channel Permeation. III. Anomalous Proton Conductance Effects in Gramicidin

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1 N HCl solutions, fluorination of channel Trp 11, Trp- 13, or Trp 15 side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K + transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.

Hydrophilic interaction/cation-exchange chromatography for separation of cyclic peptides

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which provides unique selectivities compared to reversed-phase chromatography (RPLC) for peptide separations. Separations by HILIC/CEX are effected by linear increasing salt gradients in the presence of acetonitrile (up to 90%), which promotes hydrophilic interactions overlayed on ionic interactions with the ion-exchange matrix. In the present study, the utility of HILIC/CEX has been extended to the separation of cyclic peptides in the form of synthetic model analogues of gramicidin S: Series 1 comprised six 10-residue cyclic peptide analogues which exhibited amphipathic, rigid β-pleated sheet conformation; Series 2 comprised 14-residue cyclic diastereomeric analogues of gramicidin S, where only the enantiomeric configuration of a single amino acid side-chain is varied from peptide to peptide. Observation of the retention behaviour of these two series of cyclic peptides during HILIC/CEX and RPLC confirmed not only the excellent complementarity of these two chromatographic modes but also highlighted the dramatic separations achievable by the mixed-mode approach.

Interaction and orientation of an alpha-aminoisobutyric acid- and tryptophan-containing short helical peptide pore-former in phospholipid vesicles, as revealed by fluorescence spectroscopy.

The interaction and orientation of a membrane protein ion channel model, an alpha-aminoisobutyric acid analogue of gramicidin B (GBA), in egg yolk phosphatidylcholine vesicles was studied by means of fluorescence spectroscopic techniques. GBA helices form stable ion-conducting pores in membranes [Jelokhani-Niaraki et al. (1995) J. Chem. Soc. Perkin Trans. 2, 801-808]. In an alpha-helical model for the peptide, all Trp residues (intrinsic fluorophores) are distributed near the C-terminus. Fluorescence quenching experiments revealed the exposure of the helical peptides' C-termini to aqueous environments. Dansyl-labeled vesicles were used to investigate the GBA dynamism of the interaction with membranes. It was shown that considerable amounts of peptide reside on and in the vicinity of the outer surface of lipid bilayers. The transmembrane transfer to the inner layer is slow due to the high affinity of Trp residues for bilayer interfaces which anchor the peptide to the outer surface. A structural-functional interpretation of the GBA interaction with membranes is presented.

Raman linear intensity difference of membrane-bound peptides: indole ring orientations of tryptophans 11 and 13 in the gramicidin A transmembrane channel.

A Raman linear intensity difference (RLID) method has been developed to determine orientations of chromophores in membrane-bound peptides and proteins. The method involves orientation of the peptide or protein in lipid bilayer membranes and measurement of intensity differences between Raman spectra excited with two orthogonal laser polarizations. Analysis of the RLID spectrum is simplified when the chromophore exhibits a vibrational mode for which the Raman band is enhanced through resonance with a single molecular electronic transition. To examine the indole ring orientations of Trp residues in the gramicidin A transmembrane channel, we have prepared analogues of gramicidin A, in which one of four Trp residues is replaced by deuterated Trp (Trp-2,4,5,6,7-d5). Two vibrational Raman bands omega(d)3 and omega(d)2 of deuterated Trp have been shown to gain intensity predominantly through resonance with the Bb and La electronic transitions, respectively, when excited at 244 and 257 nm. By examining the RLID spectra of the omega(d)3 and omega(d)2 bands of gramicidin A channels oriented in phospholipid bilayer membranes, we have determined the inclination angles of the Bb and La transition moments with respect to the channel axis in the absence and presence of Na+. The orientations of the Trp-11 and Trp-13 indole rings in the gramicidin channel structure have been derived from the inclination angles of the transition moments. The indole rings of Trp-11 and Trp-13, which are known to shift along the channel axis upon binding of Na+, do not reorient during their positional shifts.

Voltage-dependent behavior of a "ball-and-chain" gramicidin channel

The channel-forming properties of two analogs of gramicidin, gramicidin-ethylenediamine (gram-EDA), and gramicidin-N,N-dimethylethylenediamine (gram-DMEDA) were studied in planar lipid bilayers, using protons as the permeant ion. These peptides have positively charged amino groups tethered to their C-terminal ends via a linker containing a carbamate group. Gram-DMEDA has two extra methyl groups attached to the terminal amino group, making it a bulkier derivative. The carbamate groups undergo thermal cis-trans isomerization on the 10-100-ms time scale. The conductance behavior of gram-EDA is found to be markedly voltage dependent, whereas the behavior of gram-DMEDA is not. In addition, voltage affects the cis-trans ratios of the carbamate groups of gram-EDA, but not those of gram-DMEDA. A model is proposed to account for these observations, in which voltage can promote the binding of the terminal amino group of gram-EDA to the pore in a "ball-and-chain" fashion. The bulkiness of the gram-DMEDA derivative prevents this binding.

HPLC in the characterisation of conformational species of linear gramicidins

High-performance size-exclusion chromatography (HPSEC) has proved to be a highly simplified and rapid procedure to investigate the conformational behaviour of gramicidin A inserted in different model membrane systems (lipid dispersions, liposomes, micelles) based on the separation of double-stranded dimers and monomers present in the lipid assemblies. The HPSEC approach has been extended to the characterisation of acylated analogs of gramicidin (palmitoyl- and oleoyl-derivatives) and a series of peptide analogs where tryptophan residues were chemically modified or replaced by less polar residues (phenylalanine or naphtylalanine) lacking H-bonding ability. The Chromatographic results indicate that, in fresh preparations, the acylation modifies the dimer/monomer relationship also depending on the peptide/lipid molar ratio and membrane composition. The alteration of tryptophan residues either by chemical modification or substitution by more hydrophobic amino acids causes an appearance of double-stranded dimers, more significant as the number of tryptophan-substitutions increases and is also influenced by the specific location of the tryptophan residues in the sequence.

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.

A ProtonT1,T2, and NOE Study of Relative Motion of the Indole Ring of Tryptophans in Gramicidin Analogs Incorporated into SDS Micelles

The proton spin–lattice relaxation time, spin–spin relaxation time, and NOE of the indole ring NH proton of tryptophan residues have been determined for seven analogs of gramicidin A incorporated into SDS micelles. The data obtained indicate that the motion of the indole rings systematically decreases, proceeding from the aqueous interface to the interior of the micelle.

Gramicidin S is active against both gram‐positive and gram‐negative bacteria

Four linear and four cyclic analogs of gramicidin S (GS) in which D-Phe was replaced with either D-His, D-Ser, D-Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 micrograms/mL for gram-negative bacteria, compared to MICs of 3 micrograms/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the D-Phe to D-Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes.

Properties of synthetic analogs of gramicidin S containing L-serine or L-glutamic acid residue in place of L-ornithine residue.

In order to investigate the biological role of the delta-amino groups of the Orn residue in gramicidin S, the four analogs, [Ser2]-, [Ser2.2']-, [Glu2]- and [Glu2.2']-gramicidin S were synthesized by a solution method. Except for the last one, these analogs show antibiotic activity to a certain extent against gram-positive micro-organisms, but the activites are weaker than that of gramicidin S. These results indicate that the delta-amino group of the Orn residue is quite important for exhibiting of the activity of gramicidin S, but is not essential to the activity. NMR and CD studies of these analogs indicate that these analogs possess four intramolecular hydrogen bonds between the Val and Leu residues similar to those of GS, and the removal or diminution of the delta-amino group of ornithine residues give the considerable influence to the dihedral angle of NxH-CxH of D-Phe residues and consequently affect the type II' beta-turn structure of the cyclic peptides.

Chromatographic purification and characterization of synthetic tryptophan-substituted gramicidin A analogues

We report here experimental conditions for the one-step preparative purification of synthetic gramicidin A and 19 tryptophan-substituted analogues (with either phenylalanine or tyrosine residues) using reversed-phase chromatography. Methanol-water based mobile phases have been shown suitable for the elution of gramicidin analogues with one and two Trp→Phe substitutions, while addition of isopropanol was required for the purification of peptides with more than two Trp→Phe replacements. Using analytical RP-HPLC it is demonstrated that peptide retention increases as a monotonic function of the number of Trp→Phe substitutions, and that the position of a given amino acid substitution has little influence on peptide retention. All purified synthetic peptides give a clean, sharp characteristic peak which indicates the effectiveness of the purification protocol used. Size-exclusion HPLC reveals that purified Trp-substituted gramicidins exhibit a dimer/monomer conformational equilibrium in organic solvents similar to that observed for the naturally occurring gramicidin mixture. The usefulness of this series of Trp-substituted gramicidins in conformational studies of gramicidin incorporated in model membranes is also considered.

Species heterogeneity of Gly-11 gramicidin A incorporated into sodium dodecyl sulfate micelles

Evidence is presented for species heterogeneity of the gly-11 analog of gramicidin A incorporated into sodium dodecyl sulfate (SDS) micelles. The evidence for species heterogeneity has been obtained using one-dimensional (1D) 1H NMR spectroscopy. The 1D spectra of the indole NH moiety of tryptophans 9, 13, and 15 show the presence of more than one species. It has been found that the heterogeneity is dependent upon the gly-11/SDS molar ratio. At high SDS concentration (i.e., gly-11/SDS of 3 mM/700 mM) the heterogeneity almost completely disappears. The temperature dependence of these 1H NMR signals suggests that the two species do not interconvert. The results of nuclear Overhauser effect spectroscopy NMR experiments indicate that one species is embedded within the micelle, while the other is nearer the aqueous interface. The importance of side chain interactions with the membrane environment in producing stable, solubilized species of small peptides in SDS micelles is illustrated.

Gramicidin tryptophans mediate formamidinium-induced channel stabilization

Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl.

HPLC Demonstrates That an All Trp→Phe Replacement in Gramicidin A Results in a Conformational Rearrangement from β-Helical Monomer to Double-Stranded Dimer in Model Membranes

We have taken advantage of of previously reported high performance liquid chromatographic (HPLC) strategy to investigate the conformational behavior of the optically reversed gramicidin M (gM−), an analog of gramicidin A with all tryptophans replaced by phenylalanines, in different model membranes. It is quantitatively demonstrated for the first time that once inserted in the lipid environment, gM− (unlike the native peptide) undergoes a conformational transition from β-helical monomers to thermodynamically stable double-stranded dimers. This transition is faster the higher the incubation temperature and can be neatly observed in both small unilamellar phospholipid vesicles and lysophospholipid micelles. The results of this study are discussed in tile light of presently available data from other techniques, in the framework of the current efforts to understand structure-function relationships of linear gramicidins.

Conformational studies and pore-forming properties of an α-aminoisobutyric acid analogue of gramicidin B

Structure-function relation of a designed α-aminoisobutyric acid (Aib) analogue of gramicidin B (GBA), a model for helical protein structures, is examined by CD and patch-clamp experiments. This 16-residue peptide adopts stable helical structures in organic and aqueous solvent sytems, and phospholipid vesicles. The content of the helical structure in egg yolk phosphatidylcholine vesicles increases with the increase of lipid-to-peptide molar ratio, suggesting adsorption and incorporation processes. A possible helix–helix interaction is observable at low peptide-to-lipid molar ratios. The role of Trp side-chains for the rather high affinity of this peptide for membrane surfaces is stressed. Though shorter than the average thickness of phospholipid bilayers, GBA forms voltage-dependent multi-state ion-conducting pores in diphytanoyl phosphatidylcholine bilayers formed at the tip of micropipettes, at relatively low concentrations. A range of GBA conductances is comparable to membrane protein channels. GBA pores show a variety of conducting behaviours as well as rectifying properties.