Showing papers by "Sergii Afonin published in 2007"

The ability of Aneurinibacillus migulanus (Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotype variation.

Abstract: Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation for Aneurinibacillus migulanus (formerly known as Bacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999(T) contains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895(T) (=ATCC 9999(T)), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.

Evaluating the amino acid CF3-bicyclopentylglycine as a new label for solid-state 19 F-NMR structure analysis of membrane-bound peptides.

Abstract: The conformation, alignment and dynamic behavior of membrane-bound peptides is readily accessible by solid-state (19)F-NMR spectroscopy, but it has been difficult to incorporate suitable (19)F-labelled amino acids into synthetic peptides. To avoid the drawbacks of previously used labels, we have rationally designed and synthesized a novel amino acid that suits all theoretical and practical requirements for peptide synthesis and subsequent (19)F-NMR structure analysis [Mikhailiuk et. al, Angew. Chem. 2006, 118, 5787-5789]. The enantiomerically pure L-form of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF(3)-Bpg) carries a CF(3) group that is rigidly attached to the peptide backbone and does not racemize during peptide synthesis. It could be demonstrated for several different peptides that their biological activity is usually not affected by a single label, nor the conformation, as monitored by circular dichroism. Here, we carry out a more detailed structure analysis to evaluate the potential and reliability of CF(3)-Bpg for solid-state NMR, using the well-known alpha-helical antimicrobial peptide PGLa as a test case. We have collected several orientational constraints from the anisotropic (19)F--(19)F dipolar couplings of CF(3)-Bpg in various positions of PGLa embedded in lipid bilayers. These resulting structural parameters are then compared with those previously determined from 4-CF(3)-phenylglycine and 3,3,3-d(3)-alanine labels on the same peptide. The analysis confirms that CF(3)-Bpg does not perturb the alpha-helical conformation of PGLa. Likewise, the helix alignment is shown to follow the established concentration-dependent pattern in realigning from a surface-bound S-state to an obliquely tilted T-state. Hence, the advantages of CF(3)-Bpg over all previously used (19)F-labeled side chains are evident, as they combine ease of chemical incorporation and peptide purification with high NMR sensitivity and absent background signals, allowing a straightforward analysis of the dipolar splittings with no need for chemical shift referencing without any ambiguity in the sign of the couplings.

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

Abstract: It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF3-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity.

Using fluorinated amino acids for structure analysis of membrane-active peptides by solid-state 19F-NMR

Abstract: Several different membrane-active peptides were labeled with a variety of fluorinated amino acids for structure analysis by solid state 19 F NMR. Namely, 4-F-Phg/4-CF 3 -Phg, 3,3,3-F 3 -Ala/3-F-Ala, and 2-CF 3 -Ala were used to replace a single amino acid such as Ile/Leu, Ala, and Aib, respectively, without significantly perturbing the peptide conformation or function. These NMR reporter groups can be analyzed to calculate the structure and mobility of the peptide in the lipid bilayer. This review focuses on synthetic challenges with 19 F-labeled amino acids, such as racemization and fluorine elimination, and recent results on various antimicrobial and fusogenic peptides in model membranes will be summarized.

Using Fluorinated Amino Acids for Structure Analysis of Membrane‐Active Peptides by Solid‐State 19F‐NMR

Abstract: Several different membrane-active peptides were labeled with a variety of fluorinated amino acids for structure analysis by solid state 19 F NMR. Namely, 4-F-Phg/4-CF 3 -Phg, 3,3,3-F 3 -Ala/3-F-Ala, and 2-CF 3 -Ala were used to replace a single amino acid such as Ile/Leu, Ala, and Aib, respectively, without significantly perturbing the peptide conformation or function. These NMR reporter groups can be analyzed to calculate the structure and mobility of the peptide in the lipid bilayer. This review focuses on synthetic challenges with 19 F-labeled amino acids, such as racemization and fluorine elimination, and recent results on various antimicrobial and fusogenic peptides in model membranes will be summarized.