Liquid chromatography of gramicidin

Summary A simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 μm,250×4.6 mm I.D. column, maintained at 50°C. The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min⁻¹. Detection was by UV at 282 nm. Valine gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures determined by liquid chromatography/ mass spectrometry. The names 10-methionine valine gramicidin C, 4-methionine valine gramicidin A, valine gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustness of the liquid chromatography method was evaluated by performing a full factorial design experiment. The method also showed good repeatability, linearity and sensitivity.     

Conformation of gramicidin-A in CTAB micellar media

Gramicidin A (gA) is a linear pentadecapeptide, which exhibits various conformations depending on the environment. The conformational behavior of gA in spherical and rod-shaped cationic micelles formed by cetyltrimethylammonium bromide (CTAB) surfactant has been studied using circular dichroism (CD) and fluorescence spectroscopy, and a probable structure of gramicidin A in CTAB media has been proposed. A CD study shows that gramicidin A assumes beta(6.3) helical structure in cationic spherical as well as rod-shaped CTAB micellar media. Modeling studies show the flexibility of the side chain conformation particularly in tryptophan-9. Study of intrinsic fluorescence of tryptophans in gramicidin A indicates three distinct environments for the four-tryptophan residues in CTAB media.     

Gramicidin A interaction at a dioleoyl phosphatidylcholine monolayer on a mercury drop electrode

A biosensor where the sensing surface is a fluid dioleyl phosphatidylcholine monolayer (DOPC) deposited on a mercury drop was used. The lipid monolayer was held in 0.1 M NaCl and a concentration of gramicidin A in the range 0-12 nM was used. Electrochemical impedance spectroscopy in the frequency range 0.1-65 kHz was employed to investigate how the defect-free monolayer responds to interactions of gramicidin A in solution.The data was analyzed both with multivariate data analysis and classical electrochemical methods. The principal component analysis of the resulting impedance spectra gave a linear dependence on the concentration of gramicidin A. An increasing permittivity was observed in the low-frequency regime with increasing concentration of gramicidin A in solution.     

Gramicidin conducting dimers in lipid bilayers are stabilized by single-file ionic flux along them

Gramicidin D was incorporated in a biomimetic membrane consisting of a lipid bilayer tethered to a mercury electrode via a hydrophilic spacer, and its behavior was investigated in aqueous 0.1 M KCl by potential-step chronocoulometry and electrochemical impedance spectroscopy. The impedance spectra, recorded from 0.1 to 1 x 10(5) Hz over a potential range of 0.7 V, were fitted to a series of RC meshes, which were related to the different substructural elements of the biomimetic membrane. These impedance spectra were compared with those obtained by incorporating valinomycin, under otherwise identical conditions. The potential dependence of the stationary currents reported on bilayer lipid membranes by Bamberg and L?uger (Bamberg, E.; L?uger, P. J. Membrane Biol. 1973, 11, 177-194) as well as those extracted from potential-step chronocoulometric measurements was interpreted by relating the increase in gramicidin dimerization to a progressive increase in single-file K+ flux along the dimeric channels. An analogous approach was adopted in explaining the difference between the impedance spectra obtained with gramicidin D and those obtained with valinomycin. It is concluded that gramicidin has a low tendency to form dimers in the absence of ionic flux.     

Gramicidin A channel in a matrix from a semifluorinated surfactant monolayer

Gramicidin A, a polypeptide antibiotic forming transmembrane ion channels, has been incorporated into a Langmuir monolayer formed by a semifluorinated alkane (SFA). In this work, partially fluorinated tetracosane, perfluorohexyloctadecane (F6H18), has been applied, aiming at finding a suitable matrix for gramicidin A to be transferred onto solid support for a biosensor design. For this purpose, the physiological conditions were of special interest (mixed monolayers containing low gramicidin proportion and the surface pressure of 30 mN/m). Mixed monolayers of gramicidin and SFA were found to be miscible within the whole range of mole fractions. A very significant increase of the stability of SFA monolayer has been found in the presence of gramicidin, even at such a low proportion as X(gramicidin) = 0.1, which is reflected in a 3.5-fold increase of the collapse pressure value of mixed monolayer as compared to the film from pure SFA. This interesting phenomenon has been interpreted as being due to the existence of a strong dipole-dipole interaction between both film-forming molecules. Opposite sign of the measured electric surface potential for gramicidin and SFA, resulting from different directions of the dipole moment vectors in both film molecules, implies that the ordered, antiparallel orientation of the dipole moments in the mixed gramicidin/SFA system can be responsible for its extremely high stability.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

Structure and phase behavior of archaeal lipid monolayers

We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air-water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 ? was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only.     

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.     

Wavelength-selective fluorescence in ion channels formed by gramicidin A in membranes

Gramicidins are linear peptides that form ion channels that are specific for monovalent cations in membranes. The tryptophan residues in the gramicidin channel play a crucial role in the organization and function of the channel. The natural mixture of gramicidins, denoted as gramicidin A’, consists of mostly gramicidin A, but also contains gramicidins B, C and D as minor components. We have previously shown that the tryptophan residues in ion channels formed by the naturally occurring peptide, gramicidin A’, display wavelength-dependent fluorescence characteristics due to the motionally restricted environment in which they are localized. In order to check the influence of ground-state heterogeneity in the observed wavelength-selective fluorescence of gramicidin A’ in membranes, we performed similar experiments with pure gramicidin A in model membranes. Our results show that the observed wavelength-selective fluorescence characteristics of naturally occurring gramicidin A’ are not due to ground-state heterogeneity.     

A practical synthesis of gramicidin s and sugar amino Acid containing analogues

A practical gram-scale and high-yielding synthesis of the antimicrobial peptide gramicidin S is presented. An Fmoc-based solid-phase peptide synthesis protocol is employed for the generation of the linear decapeptide precursor, which is cyclized in solution to afford the target compound. The versatility of our method is demonstrated by the construction of eight gramicidin S analogues (15a-h) having nonproteinogenic sugar amino acid residues (4-7) incorporated in the turn regions.     

Conformation and Interaction of a D,L-alternating peptide with a bilayer membrane: x-ray reflectivity, CD, and FTIR spectroscopy.

Peptides with alternating amino acid configuration provide helical secondary structures that are especially known from the membrane channel and pore-forming gramicidin A. In analogy to this natural D,L-alternating pentadecapeptide, the potential of D,L-alternating peptides for membrane insertion is investigated using the model dodecamer peptide H-(Phe-Tyr)(5)-Trp-Trp-OH. This aromatic peptide is introduced as a novel pore-forming synthetic analogue of gramicidin A. It forms a well-organized homodimer similar to one of the gramicidin A transmembrane motifs. X-ray reflectivity measurements are performed on solid-supported peptide-lipid complexes to obtain information about the influence of the artificial dodecamer peptide on the bilayer parameters. In addition, Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopic studies determine the conformational state of H-(Phe-Tyr)(5)-Trp-Trp-OH within the model membrane. Site-specific iodine labeling assists in determining the topology of the membrane-embedded peptide by pinpointing the position of the iodine label within the bilayers.     

Separation of linear gramicidins using carbon dioxide-containing mobile phases

Packed-column supercritical-fluid chromatography (pSFC) is presented as a novel method for separating and analyzing gramicidin samples. By use of methanol-modified carbon dioxide as a mobile phase the pentadecapeptides gramicidin A (gA), gramicidin B (gB), and gramicidin C (gC) are readily separated and eluted from a PRP-1 poly(styrene–divinylbenzene) column. Although optimum separation conditions are typically achieved near a column temperature of 40°C, a column pressure of 11 MPa, and 30% methanol modifier, pressure and modifier gradients around these values are also found to improve the overall separation time. Measurements indicate that the mobile phase solubility of gramicidin under these conditions is 5.0±0.4 g mL^–1. Collection of individual peaks during chromatography achieved analytical-scale isolation of 2 g refined gC from 20 g injected gramicidin D. Further, supercritical-fluid extraction of 200 g gramicidin D from a Chromosorb 102 support packed into the vessel produced 57 g gA in 90% purity. The results establish that carbon dioxide-based mobile phases can be successfully used for the separation of individual gramicidin species.     

Ion mobility-mass spectrometry applied to cyclic peptide analysis: Conformational preferences of gramicidin S and linear analogs in the gas phase

In this paper, we present an investigation of the gas-phase structural differences between cyclic and linear peptide ions by matrix-assisted laser desorption ionization-ion mobility-mass spectrometry. Specifically, data is shown for gramicidin S (cyclo-VOLFPVOLFP where phenylalanines are D rather than L-type amino acids and the O designates the non-standard amino acid ornithine) and five linear gramicidin S analogues. Results are interpreted as evidence for a beta-sheet (or beta-hairpin) conformational preference in both linear-protonated and sodiated-cyclic gramicidin S gas-phase peptides, and a preference for the protonated-cyclic peptide to adopt a collapsed, random coil-type conformation. A comparison with solution-phase circular dichroism measurements is performed, and structures similar to those observed in the gas phase appear to be favored in low-dielectric solvents such as 2,2,2-triflouroethanol. The utility of ion mobility-mass spectrometry (IM-MS) as a means of rapidly distinguishing between linear and cyclic peptide forms in also discussed.     

Phosphatidylcholine-Gramicidin a Interaction Study in Non-Aqueous Solvent with a new HPLC-SEC Column

Abstract

A new HPLC-SEC column, Ultrastyragel 500 Å, has been assayed in order to check the separation between gramicidin A and phosphatidylcholine in tetrahydrofuran. The good resolution of both compounds has allowed the quantitation of lipid-polypeptide interaction in terms of the binding ratio parameter, BR. A BR value of 3.6 moles of phosphatidylcholine per mole of gramicidin A has been obtained for a 0.1 % (w/v) phosphatidylcholine solution as eluent. The suitability of this support for studies on lipid-polypeptide interactions in non-aqueous solvents is demonstrated.     

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.     

15N,(1)H Heteronuclear correlation NMR of gramicidin A in DMPC-d(67)

We have studied gramicidin A, an environmentally sensitive polymorphic pentadecapeptide, fully 15N-labelled and dispersed in a highly deuterated phospholipid bilayer system. By submitting the sample to fast magic angle spinning, we were able to reduce the polypeptide amide hydrogen linewidths to 160 Hz, and hence to partially resolve them. By correlating these resonances with the 40 Hz wide dipolar coupled 15N in a 2D-CROPSY (cross-polarization spectroscopy) experiment, it was possible to observe the 20 partially overlapping 1H-15N signal pairs from the polypeptide backbone and sidechains. Both chemical shift distributions closely match those of the same peptide in SDS micelles, but only poorly match those of conformationally different gramicidin A in trifluoroethanol, dimethylsulfoxide, or methanol/chloroform mixture. Our results are indicative of the N-to-N right-handed beta6.3-helix conformation of gramicidin A and offer sufficient resolution to encourage development of experiments to measure orientational or distance restraints using through-space dipolar couplings.     

Photopolymerization of mixed monolayers and black lipid membranes containing gramicidin A and diacetylenic phospholipids

We formed monolayers and black lipid membranes (BLMs) of photopolymerizable lipids mixed with the channel-forming protein gramicidin A to evaluate their miscibility and the potential for improved stability of the BLM scaffold through polymerization. Analyses of surface pressure vs area isotherms indicated that gramicidin A dispersed with three different synthetic, polymerizable, diacetylene-containing phospholipids, 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE), and 1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (PTPE) to form mixed monolayers at the air-water interface on a Langmuir-Blodgett (LB) trough. Conductance measurements across a diacetylenic lipid-containing BLM confirmed dispersion of the gramicidin channel with the lipid layer and demonstrated gramicidin ion-channel activity before and after UV exposure. Polymerization kinetics of the diacetylenic films were monitored by film pressure changes at constant LB trough area and by UV-vis absorption spectroscopy of polymerized monolayers deposited onto quartz. An initial increase in film pressure of both the pure diacetylene lipid monolayers and mixed films upon exposure to UV light indicated a change in the film structure. Over the time scale of the pressure increase, an absorbance peak indicative of polymerization evolved, suggesting that the structural change in the lipid monolayer was due to polymerization. Film pressure and absorbance kinetics also revealed degradation of the polymerized chains at long exposure times, indicating an optimum time of UV irradiation for maximized polymerization in the lipid layer. Accordingly, exposure of polymerizable lipid-containing black lipid membranes to short increments of UV light led to an increase in the bilayer lifetime.     

Self-assembled-monolayer-modified silicon substrate to enhance the sensitivity of peptide detection for AP-MALDI mass spectrometry

A self-assembled-monolayer-modified silicon substrate was successfully used to enhance the sensitivity of peptide detection for atmospheric pressure-matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI/MS). The effect of surface modification of silicon wafer samples with NH(2) and OH functional groups was investigated. In addition, solvent effects for the preparation of modified NH(2)-functionalized surfaces were examined. The sensitivities for the two peptides were significantly improved, increasing between 12 and 160 times, for bradykinin and gramicidin, respectively, on an NH(2)-modified silicon surface prepared in toluene, over that on a conventional gold substrate. The limits of detection (LODs) for bradykinin and gramicidin using the conventional gold substrate in AP-MALDI/MS experiments were > 0.011 microM and 110 microM, respectively. Using our SAM approach, the LODs for bradykinin and gramicidin in AP-MALDI/MS can be improved to 0.93 nM and 0.33 microM, respectively. This SAM approach for AP-MALDI/MS is simple and sensitive, and can be used for high-throughput analysis.     

The effect of interaction between K(+) ions and gramicidin D on the lecithin membrane interfacial tension

The effect of the presence of gramicidin D in a lecithin membrane on its interfacial tension has been studied. The studies have been carried out at various forming solution compositions and at various potassium ion concentrations in the electrolyte solution. Potassium chloride was used as the electrolyte. The complex was formed between the gramicidin molecule and K(+) ion. The following parameters describing the complex were determined: the surface area occupied by GK(+) complex (A(GK(+))), the interfacial tension of the GK(+) membrane complex (gamma(GK+)), and the stability constant of the gramicidin-K(+) complex (K). These values are 156 A(2), 1.89 mN m(-1) and 0.033 m(3) mol(-1), respectively.