The conformation of gramicidin S and its N,N′-diacetyl derivative in solutions

Ovchinnikov, Yu.A.; Иванов, В. Т.; Bystrov, V. F.; Мирошников, А. И.; Shepel, E.N.; Абдуллаев, Н. Д.; Efremov, E. S.; Senyavina, L. B.

doi:10.1016/0006-291x(70)90781-3

Cited by 80 publications

(29 citation statements)

References 26 publications

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“…Surprisingly, GS does not readily produce X-ray quality single crystals; hence, highly artificial crystallization conditions were applied in both cases, in part explaining these discrepancies. The picture is somewhat more consistent for the NMR-derived structures that have been obtained in membrane-mimicking solutions, such as dimethyl sulfoxide (DMSO) (13) or a CHCl 3 -methanol mixture (14), but in most NMR studies GS derivatives have been used rather than real GS. Most significantly, GS is a membrane-active peptide; hence, functionally relevant structural information should be acquired when it is bound to a genuine lipid bilayer.…”

Section: T He Cyclic Decapeptide Gramicidin S (Gs) Contains a Repeat mentioning

confidence: 79%

“…The current paper is focused on the critical process of producing 13 C/ 15 N-labeled GS, whereupon a fermentation procedure was devel- (14); VII, GS in crystal, grown in the presence of urea and HCl (11); VIII, GS in crystal, grown in the presence of TFA and HCl (12); IX, free GS structure (in vacuo) as predicted in reference 9; X, free GS structure (in vacuo) as predicted in reference 10. oped. The method allows 100% labeling efficiency and is costeffective, as was desired for being practically useful.…”

Section: Discussionmentioning

confidence: 99%

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Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Berditsch

Afonin

Steineker

et al. 2015

Appl Environ Microbiol

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Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membranebound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13 C/ 15 N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13 C/ 15 N-labeled amino acids. The most cost-effective production of 13 (Fig. 1A). Since its discovery about 70 years ago, GS has been used as a topical antibiotic in Russia and neighboring countries, exploiting its pronounced antimicrobial activity against Gram-positive bacteria (1, 2). For example, GS is the bioactive agent in GrammidinNeo, the lozenge against sore throat and mouth ulcer produced by the Russian JSC Valenta Pharmaceuticals. Notably, despite a long medication history, no clinical cases of bacterial resistance against GS have been reported, which suggest that this peptide may be an exceptionally promising resistance-free antibiotic (3).The biological activity of GS was first described as membranolytic, which is believed to determine its profound antimicrobial as well as hemolytic activities in vitro. Less acknowledged are GS activities not directly related to interaction with membrane bilayers: the peptide has been shown to inhibit membrane enzymes, such as bacterial NADH dehydrogenase and cytochrome bd terminal oxidase (4), and to delocalize the peripheral cell division regulator MinD, the lipid II biosynthesis protein MurG, and cytochrome c (5).The main molecular target of GS is supposed to be the plasma membrane of susceptible bacterial species, where an accumulation of the amphiphilic peptide leads to a loss of the barrier function and an irreversible increase in permeability for ions and metabolites (6). This concentration-dependent effect leads to membrane depolarization (7) and an overall dysfunction of the cellular osmoregulation, which starts already at GS levels well below the respective MICs (8). However, the structure-function relationship of the peptide and its detailed molecular mechanism of action at the plasma membrane, i.e., its interaction with the lipid bilayer, are still lacking a consensus view.Over the last decades, the molecular structure of GS has been stud...

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Section: T He Cyclic Decapeptide Gramicidin S (Gs) Contains a Repeat mentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Berditsch

Afonin

Steineker

et al. 2015

Appl Environ Microbiol

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“…13. The Xl and X2 structures are derived from the models given by De Santis and Liquori (25), and the X3 structure is derived from the model of Ovchinnikov et al (26). Examination of the R factors** for these structures shows that these are as successful as the Ml-M3 structures in explaining the NOE data.…”

Section: Resultsmentioning

confidence: 99%

Solution conformation of gramicidin S: An intramolecular nuclear Overhauser effect study

Huang

Walter

Glickson

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The solution conformation of gramicidin S in deuterated dimethyl sulfoxide was investigated by using the intramolecular nuclear Overhauser effect experiment. Experimental Overhauser enhancements were compared with predicted values for each ofthe nine most-stable conformations (M1-M9) calculated by Dygert et al on the basis of energy-minimization procedures [Dygert, M., Go, N. & Scheraga, H. A. (1975) Macromolecules 8,[750][751][752][753][754][755][756][757][758][759][760][761]. By using statistical hypothesis testing, the three lowestenergy conformations (MI, M2, and M3) were shown to give the best fit with the experimental data. All other conformations (M4-M9) were found to be inconsistent with the experimental data.Intramolecular nuclear Overhauser effects (NOEs) are very sensitive to interproton distances when dipole-dipole interaction is the predominant mechanism ofproton relaxation (1). Because of this distance dependence, NOE measurements have been used extensively in conformational studies of small molecules (1); however, their application to peptides is relatively recent (2-7).The cyclic decapeptide antibiotic gramicidin S has been the subject ofmany experimental [ref. 8 perature was 297 ± 1 K. In the NOE experiment, the resonance signal of interest was selectively presaturated for 4 sec before the observation pulse. The decoupler power was set such that the irradiation was highly selective; the decoupler power spillover to the neighboring resonance signals was less than 2% in general, as determined from the difference spectra. For the control spectrum, the irradiation frequency was set away from the spectral region by more than 2 kHz. The degree of saturation of the irradiated lines was 80-90%, and the observed NOEs were finally corrected for 100% saturation of the irradiated peak by dividing by the degree of saturation. One thousand scans were accumulated for each spectrum. To minimize the effects associated with spectrometer drift and to obtain clean difference spectra, the accumulation was alternated every eight scans between the control and the NOE experiments. A 0.5-Hz exponential line broadening was used in Fourier transforming the free induction decays. The NOE difference spectra were obtained by subtracting the control spectrum from the double resonance spectra. The NOE enhancements were calculated as fractional intensity changes for each resonance signal. For overlapping signals and for the C'H2 multiplet of D-phenylalanine, integrated intensities were used in computing the NOEs. RESULTS AND DISCUSSIONThe nuclear Overhauser effect experiment measures the steadystate effect of saturation of a specific spin transition or group of transitions on the intensities of other resonances in the NMR spectrum. The fractional change in the intensity of spin i when a spin s (or a group of such spins) is saturated can be computed by solving a set ofsimultaneous equations of the form (2) oijff(s) + I oyfj(s)where the summation on the right-hand side involves only the saturated spins. The oii and (ri...

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“…It has recently become evident that nmr spectroscopy will be of use in determining conformations of cyclic polypeptides (1)(2)(3)(4)(5)(6)(7)(8). In particular, we have been interested in conformationactivity correlations in a series of neurohypophyseal hormones, including oxytocin and its derivatives.…”

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confidence: 99%

NMR Studies on the Conformation of Derivatives of the Side Chain of Oxytocin: Examples of cis-trans Isomerism

Hruby

Brewster

Glasel

1971

Proc. Natl. Acad. Sci. U.S.A.

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The 220 MHz spectra reported in this paper show the existence of cis-trans isomerism about the Cys-Pro bond in (S-benzyl)-L-Cys-L-Pro-L-Leu-Gly(NH2) and about the Z-Pro bond in (N-benzyloxycarbonyl)-LPro-L-Leu-Gly(NH2). These peptides are derivatives of the side chain of oxytocin, which has the structure L-Pro-LLeu-Gly(NH2). Taken in water-free (CDa)2SO, the spectra also show differences between the two isomers in the amide chemical shifts, which indicate interaction of the terminal end of the peptides with the rest of the residues.

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The conformation of gramicidin S and its N,N′-diacetyl derivative in solutions

Cited by 80 publications

References 26 publications

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Solution conformation of gramicidin S: An intramolecular nuclear Overhauser effect study

NMR Studies on the Conformation of Derivatives of the Side Chain of Oxytocin: Examples of cis-trans Isomerism

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The conformation of gramicidin S and its N,N′-diacetyl derivative in solutions

Cited by 80 publications

References 26 publications

Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Solution conformation of gramicidin S: An intramolecular nuclear Overhauser effect study

NMR Studies on the Conformation of Derivatives of the Side Chain of Oxytocin: Examples of cis-trans Isomerism

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Product

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Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis