Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

The first non-helical Aib-containing hexapeptide: The crystal structure of Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu was crystallized from a mixture of ethyl acetate and n-hexane. The crystals belong to the centrosymmetric space group Pbca. There are three molecules in the asymmetric unit. The three molecules differ mainly in the Z-group conformation. The first Gly residue adopts a fully extended conformation, residues 2 and 3 lie in the left-handed helical region, residues 4 and 5 in the right-handed helical region, and residue 6 again in the left-handed helical region of the Ramachandran plot. There are only two of four possible intramolecular hydrogen bonds formed, namely, between Aib4 and Gly1 forming a ß-turn of type III' and between Aib6 and Gly3 forming a ß-turn of type I. The inverted molecules (by space group symmetry) lie in the regions with opposite handedness and form ß-turns of type III and I'. In contrast to all known long synthetic and naturally occurring Aib-containing peptides that fold as 310 - or α-helix, Z-(Gly-Aib)3 -OtBu folds in a quite flat structure from which only the protecting groups bulge out.

Removing the C-terminal protecting group enlarges the crystal size: Z-(Gly-Aib)2-OH·H2O.

The achiral tetrapeptide monohydrate N-(benzyloxycarbonyl)glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid monohydrate, Z-Gly-Aib-Gly-Aib-OH·H2O (Z is benzyloxycarbonyl, Aib is α-aminoisobutyric acid and Gly is glycine) or C20H28N4O7·H2O, exhibits two conformations related by the symmetry operation of an inversion centre. It adopts only one of two possible intramolecular hydrogen bonds in a type I (and I') ß-turn and forms a maximum of intermolecular hydrogen bonds partly mediated by water. The space group, the molecular structure and the crystal packing differ from two already described (Gly-Aib)2 peptides which vary only in the protecting groups. This structure confirms the high structural flexibility of Gly-Aib peptides and points to a strong relationship between intermolecular hydrogen bonding and crystal quality and size.

Sequences of Tolypins, Insecticidal Efrapeptin-Type Peptaibiotics from Species of the Fungal Genus Tolypocladium.

A peptide mixture named tolypin, originally isolated from species of the fungal genus Tolypocladium, was structurally characterised and sequences compared to those reported for efrapeptins isolated from strains of Tolypocladium inflatum. Chiral amino acid analysis, direct infusion, and online HPLC electrospray ionization tandem mass spectrometry provided composition, molecular weights of peptides, and series of diagnostic fragment ions. Sequences deduced from ESI-MS revealed that tolypins C-G are identical to efrapeptins C-G. The results were corroborated by ESI-MS and HPLC of an authentic efrapeptin sample from Eli Lilly Research Laboratories (USA). Comparison of the HPLC elution profiles of efrapeptin and tolypin indicated a pronounced microheterogeneity of the former. A high-resolution HPLC of authentic efrapeptin has not been published before. Close relationship and partial identity of sequences of tolypins and efrapeptins, which had previously been postulated, were definitely proven. The geographical origin of the two most important T. inflatum strains used for sequencing of efrapeptins/tolypins could unambiguously be clarified. A new minor compound, designated tolypin H1, was sequenced. High proportions of helicogenic Aib (α-aminoisobutyric acid) and l-isovaline, N-terminal acetyl-l-pipecolic acid and the unusual, amide-bound C-terminal residue, named (S)-2-amino-1-(1,5-diazabicyclo[4.3.0]non-5-ene-5-ylium)-4-methylpentane corresponding to 1-[(2S)-2-amino-4-methylpentyl]-2,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrimidin-1-ium, define these peptides as linear, cationic peptaibiotics.

Dispersion analysis of the orthorhombic peptide single crystal Z-(Aib)3-L-Ala-OtBu.

We present a dispersion analysis of the orthorhombic peptide single crystal Z-Aib-Aib-Aib-L-Ala-OtBu (N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyryl-L-alanine tert-butyl ester, C27H42N4O7), where Z is benzyloxycarbonyl and OtBu is the tert-butylester) in the MIR spectral region by means of adapted generalized dispersion analysis, employing the naturally grown crystal faces. Based on the results we identify the orientation of the crystal axes a, b, c within the sample, and supported by a stereographic projection of the crystal, we assign the individual axes. The gained dielectric tensor function and the oscillator parameters were confirmed by forward calculation of reflection spectra of different orientations. The orientation of the crystal axes was verified by a second stereographic projection with another crystal face in the center.

Crystal structures of Z-Gly-Aib-O-·0.5Ca2+·H2O and Z-Gly-Aib-OH.

Both deprotonated and neutral achiral title dipeptides assume similar structures of two conformations, which are related by a unit-cell inversion centre. Two mol-ecules of both conformations of the metal-free neutral dipeptide are linked by two hydrogen bonds, while two mol-ecules of both conformations of the ionized form coordinate a calcium ion in calcium(II) bis-[2-(2-{[(benz-yl-oxy)carbon-yl]amino}-acetamido)-2-methyl-propano-ate] monohydrate, 0.5Ca2+·C14H17N2O5-·0.5H2O, which lies on an inversion centre and forms a distorted octa-hedral complex with the metal ion. These CaII complexes are connected in the crystal via hydrogen bonds in the b- and c-axis directions, whereas in the a-axis direction, they stack via apolar contacts. In the metal-free crystal, namely 2-(2-{[(benz-yloxy)carbon-yl]amino}-acetamido)-2-methyl-propanoic acid, C14H18N2O5, mol-ecules are hydrogen bonded in the a- and c-axis directions, and stack in the b-axis direction via apolar contacts.

The crystal structure of the lipoaminopeptaibol helioferin, an antibiotic peptide from Mycogone rosea.

The crystal structure of the natural nonapeptide antibiotic helioferin has been determined and refined to 0.9 Šresolution. Helioferin consists of helioferin A and B, which contain 2-(2'-aminopropyl)aminoethanol (Apae) and 2-[(2'-aminopropyl)methylamino]ethanol (Amae) at their respective alkanolamine termini. In addition, helioferin contains the unusual amino-acid residues α-aminoisobutyric acid (Aib) and (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (Ahmod). The amino-terminus is capped with 2-methyl-n-1-octanoic acid (M8a). The peptide crystallizes with a 1:1 molar ratio of helioferin A and B in the monoclinic space group C2, with unit-cell parameters a = 34.711, b = 10.886, c = 17.150 Å, ß = 93.05°. The peptide backbone folds in a regular right-handed α-helical conformation, with eight intramolecular hydrogen bonds, all but one forming 5→1 interactions. The two aliphatic chains of the fatty-acyl (M8a) and the second residue (Ahmod) extend out of the α-helical structure in opposite directions and lead to a corkscrew-like shape of the peptide molecule. Halogen anions (Cl- and F-) have been co-crystallized with the peptide molecules, implying a positive charge at the aminoalcohol end of the peptide. In the tightly packed crystal the helices are linked head to tail via the anions by electrostatic, hydrogen-bond and van der Waals interactions, forming continuous helical rods. Two nonparallel rods (forming an angle of 118°) interact directly via hydrogen bonds and via the anions, forming a double layer. Successive double layers are held together only via van der Waals contacts. The helical axes of successive double layers are also related by an angle of 118°. The structure of helioferin reported here and the previously determined structure of the homologous leucinostatin A have a total straight length of about 21 Å, indicating a different membrane-modifying bioactivity from that of long-chain, amphiphilic peptaibols.

Synergistic combination of alkylphosphocholines with peptaibols in targeting Leishmania infantum in vitro.

Anti-leishmanial treatment increasingly encounters therapeutic limitations due to drug toxicity and development of resistance. The effort for new therapeutic strategies led us to work on combinations of chemically different compounds that could yield enhanced leishmanicidal effect. Peptaibols are a special type of antimicrobial peptides that are able to form ion channels in cell membranes and potentially affect cell viability. We assayed the antileishmanial activity of two well studied helical peptaibols, the 16-residue antiamoebin and the 20-residue alamethicin-analogue suzukacillin, and we evaluated the biological effect of their combination with the alkylphosphocholine miltefosine and its synthetic analogue TC52. The peptaibols tested exhibited only moderate antileishmanial activity, however their combination with miltefosine had a super-additive effect against the intracellular parasite (combination index 0.83 and 0.43 for antiamoebin and suzukacillin respectively). Drug combinations altered the redox stage of promastigotes, rapidly dissipated mitochondrial membrane potential and induced concatenation of mitochondrial network promoting spheroidal morphology. These results evidenced a potent and specific antileishmanial effect of the peptaibols/miltefosine combinations, achieved with significantly lower concentrations of the compounds compared to monotherapy. Furthermore, they revealed the importance of exploring novel classes of bioactive compounds such as peptaibols and demonstrated for the first time that they can act in synergy with currently used antileishmanial drugs to improve the therapeutic outcome.

A natural, single-residue substitution yields a less active peptaibiotic: the structure of bergofungin A at atomic resolution.

Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Šresolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

Sonification of Arm Movements in Stroke Rehabilitation - A Novel Approach in Neurologic Music Therapy.

Gross motor impairments are common after stroke, but efficient and motivating therapies for these impairments are scarce. We present an innovative musical sonification therapy, especially designed to retrain patients' gross motor functions. Sonification should motivate patients and provide additional sensory input informing about relative limb position. Twenty-five stroke patients were included in a clinical pre-post study and took part in the sonification training. The patients' upper extremity functions, their psychological states, and their arm movement smoothness were assessed pre and post training. Patients were randomly assigned to either of two groups. Both groups received an average of 10 days (M = 9.88; SD = 2.03; 30 min/day) of musical sonification therapy [music group (MG)] or a sham sonification movement training [control group (CG)], respectively. The only difference between the two protocols was that in the CG no sound was played back during training. In the beginning, patients explored the acoustic effects of their arm movements in space. At the end of the training, the patients played simple melodies by coordinated arm movements. The 15 patients in the MG showed significantly reduced joint pain (F = 19.96, p < 0.001) in the Fugl-Meyer assessment after training. They also reported a trend to have improved hand function in the stroke impact scale as compared to the CG. Movement smoothness at day 1, day 5, and the last day of the intervention was compared in MG patients and found to be significantly better after the therapy. Taken together, musical sonification may be a promising therapy for motor impairments after stroke, but further research is required since estimated effect sizes point to moderate treatment outcomes.

Sequences of stilboflavin C: towards the peptaibiome of the filamentous fungus Stilbella (= Trichoderma) flavipes.

Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α-aminoisobutyric acid-containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF-A, SF-B, and SF-C. Although all three of these groups resolved as single spots on thin-layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF-C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF-ESI-HRMS provided the sequences of 10 16-residue peptides and five 19-residue peptides, all of which were N-terminally acetylated. In contrast to the previously described SF-A and SF-B peptaibols, SF-C peptaibols contain Ser-Alaol or Ser-Leuol, which are rarely found as C-termini, and repetitive Leu-Aib-Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF-A and SF-B into account, the entirety of peptides produced by S. flavipes (the 'peptaibiome') approaches or exceeds 100 non-ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The crystal structure of Z-(Aib)10-OH at 0.65 Å resolution: three complete turns of 310-helix.

The synthetic peptide Z-(Aib)10-OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub-atomic resolution, where the bonding electron density becomes visible between the non-hydrogen atoms. Crystals belong to the centrosymmetric space group P1. Both molecules in the asymmetric unit form regular 310 -helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib-homopeptides. These other peptides are C-terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C-terminal protection group to form a hydrogen bond to the residue i-3, the sense of the helix is reversed in the last residue. Here, the C-terminal OH-groups form hydrogen bonds to the residues i-3, in part mediated by water molecules. This makes Z-(Aib)10-OH an Aib-homopeptide with three complete 310-helical turns in spite of the shorter length it has compared with Z-(Aib)11-OtBu, the only homopeptide to date with three complete turns.

The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

Gas chromatographic separation of stereoisomers of non-protein amino acids on modified γ-cyclodextrin stationary phase.

Stereoisomers (enantiomers and diastereoisomers) of synthetic, non-protein amino acids comprising α-, ß-, and γ-amino acids, including α,α-dialkyl amino acids, were converted into the respective N-trifluoroacetyl-O-methyl esters and analyzed and resolved by gas chromatography (GC) on a commercial fused silica capillary column coated with the chiral stationary phase octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. This column is marketed under the trade name Lipodex(®) E. Chromatograms, retention times, and a chart displaying the retention times of approximately 40 stereoisomers of amino acids are presented. With few exceptions, baseline or almost baseline resolution was achieved for enantiomers and diastereoisomers. The chromatographic method presented is considered to be highly suitable for the elucidation of the stereochemistry of non-protein amino acids, for example in natural products, and for evaluating the enantiopurity of genetically non-coded amino acids used for the synthesis and design of conformationally tailored peptides. The method is applicable to extraterrestrial materials or can be used in experimental work related to abiotic syntheses or enantioselective destruction and amplification of amino acids.

The peptaibiotics database--a comprehensive online resource.

In this work, we present the 'Peptaibiotics Database' (PDB), a comprehensive online resource, which intends to cover all Aib-containing non-ribosomal fungal peptides currently described in scientific literature. This database shall extend and update the recently published 'Comprehensive Peptaibiotics Database' and currently consists of 1,297 peptaibiotic sequences. In a literature survey, a total of 235 peptaibiotic sequences published between January 2013 and June 2014 have been compiled, and added to the list of 1,062 peptides in the recently published 'Comprehensive Peptaibiotics Database'. The presented database is intended as a public resource freely accessible to the scientific community at peptaibiotics-database.boku.ac.at. The search options of the previously published repository and the presentation of sequence motif searches have been extended significantly. All of the available search options can be combined to create complex database queries. As a public repository, the presented database enables the easy upload of new peptaibiotic sequences or the correction of existing informations. In addition, an administrative interface for maintenance of the content of the database has been implemented, and the design of the database can be easily extended to store additional information to accommodate future needs of the 'peptaibiomics community'.

Peptaibol, secondary-metabolite, and hydrophobin pattern of commercial biocontrol agents formulated with species of the Trichoderma harzianum complex.

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant-protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna(®) , Trichosan(®) , Vitalin(®) , Promot(®) WP, and TrichoMax(®) , formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well-established, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma- based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

The crystal structure of Z-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petroleum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Structural Database. The first Gly residue assumes a semi-extended conformation (φ ±62°, ψ ∓131°). The right-handed peptide folds in two consecutive ß-turns of type II' and type I or an incipient 310 -helix, and the left-handed counterpart folds accordingly in the opposite configuration. In the crystal lattice, one molecule is linked to four neighbors in the ab-plane via hydrogen bonds. These bonds form a continuous network of left- and right-handed molecules. The successive ab-planes stack via apolar contacts in the c-direction. An ethyl acetate molecule is situated on and close to the fourfold axis.

Taurine is absent from amino components in fruits of Opuntia ficus-indica.

Juices of edible fruits from Opuntia ficus-indica (L.) Miller, commonly named prickly pears or Indian figs, were analysed for amino acids using an automated amino acid analyser run in the high-resolution physiological mode. Emphasis was put on the detection of free taurine (Tau), but Tau could be detected neither in different cultivars of prickly pears from Italy, South Africa and the Near East nor in commercially available prickly pear juices from the market.

The achiral tetrapeptide Z-Aib-Aib-Aib-Gly-OtBu.

The title achiral peptide N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyrylglycine tert-butyl ester or Z-Aib-Aib-Aib-Gly-OtBu (Aib is α-aminoisobutyric acid, Z is benzyloxycarbonyl, Gly is glycine and OtBu indicates the tert-butyl ester), C26H40N4O7, is partly hydrated (0.075H2O) and has two different conformations which together constitute the asymmetric unit. Both molecules form incipient 310-helices. They differ in the relative orientation of the N-terminal protection group and at the C-terminus. There are two 4→1 intramolecular hydrogen bonds.