Time-resolved fluorescence of tryptophan characterizes membrane perturbation by cyclic lipopeptides.

Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H2O-D2O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation.

Breaking Cycles: Saponification-Enhanced NMR Fingerprint Matching for the Identification and Stereochemical Evaluation of Cyclic Lipodepsipeptides from Natural Sources.

We previously described NMR based fingerprint matching with peptide backbone resonances as a fast and reliable structural dereplication approach for Pseudomonas cyclic lipodepsipeptides (CLiPs). In combination with total synthesis of a small library of configurational CLiP congeners this also allows unambiguous determination of stereochemistry, facilitating structure-activity relationship studies and enabling three-dimensional structure determination. However, the on-resin macrocycle formation in the synthetic workflow brings considerable burden and limits universal applicability. This drawback is here removed altogether by also transforming the native CLiP into a linearized analogue by controlled saponification of the ester bond. This eliminates the need for macrocycle formation, limiting the synthesis effort to linear peptide analogues. NMR fingerprints of such linear peptide analogues display a sufficiently distinctive chemical shift fingerprint to act as effective discriminators. The approach is developed using viscosin group CLiPs and subsequently demonstrated on putisolvin, leading to a structural revision, and tanniamide from Pseudomonas ekonensis COR58, a newly isolated lipododecapeptide that defines a new group characterized by a ten-residue large macrocycle, the largest to date in the Pseudomonas CLiP portfolio. These examples demonstrate the effectiveness of the saponification- enhanced approach that broadens applicability of NMR fingerprint matching for the determination of the stereochemistry of CLiPs.

Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides.

Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 µM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 µM) than viscosin (15 µM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the "cation attacks anionic membrane" selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50.

Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in Pseudomonas Lipopeptide Biosynthesis.

In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either 'on' (active) or 'off' (inactive). Here, we report the unprecedented observation of an E/C-domain that acts 'on and off', giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures.

Nanobody Loop Mimetics Enhance Son of Sevenless 1-Catalyzed Nucleotide Exchange on RAS.

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active guanosine triphosphate (GTP)-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14 (Nb14), which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of Nb14 with an EC50 value of 29 µM. Altogether, this study demonstrated that peptides able to modulate a protein-protein interaction can be obtained by structural mimicry of a Nb paratope.

Transporter Gene-Mediated Typing for Detection and Genome Mining of Lipopeptide-Producing Pseudomonas.

Pseudomonas lipopeptides (LPs) are involved in diverse ecological functions and have biotechnological application potential associated with their antimicrobial and/or antiproliferative activities. They are synthesized by multimodular nonribosomal peptide synthetases which, together with transport and regulatory proteins, are encoded by large biosynthetic gene clusters (BGCs). These secondary metabolites are classified in distinct families based on the sequence and length of the oligopeptide and size of the macrocycle, if present. The phylogeny of PleB, the MacB-like transporter that is part of a dedicated ATP-dependent tripartite efflux system driving export of Pseudomonas LPs, revealed a strong correlation with LP chemical diversity. As each LP BGC carries its cognate pleB, PleB is suitable as a diagnostic sequence for genome mining, allowing assignment of the putative metabolite to a particular LP family. In addition, pleB proved to be a suitable target gene for an alternative PCR method for detecting LP-producing Pseudomonas sp. and did not rely on amplification of catalytic domains of the biosynthetic enzymes. Combined with amplicon sequencing, this approach enabled typing of Pseudomonas strains as potential producers of a LP belonging to one of the known LP families, underscoring its value for strain prioritization. This finding was validated by chemical characterization of known LPs from three different families secreted by novel producers isolated from the rice or maize rhizosphere, namely, the type strains of Pseudomonas fulva (putisolvin), Pseudomonas zeae (tensin), and Pseudomonas xantholysinigenes (xantholysin). In addition, a new member of the Bananamide family, prosekin, was discovered in the type strain of Pseudomonas prosekii, which is an Antarctic isolate. IMPORTANCE Pseudomonas spp. are ubiquitous bacteria able to thrive in a wide range of ecological niches, and lipopeptides often support their lifestyle but also their interaction with other micro- and macro-organisms. Therefore, the production of lipopeptides is widespread among Pseudomonas strains. Consequently, Pseudomonas lipopeptide research not only affects chemists and microbiologists but also touches a much broader audience, including biochemists, ecologists, and plant biologists. In this study, we present a reliable transporter gene-guided approach for the detection and/or typing of Pseudomonas lipopeptide producers. Indeed, it allows us to readily assess the lipopeptide diversity among sets of Pseudomonas isolates and differentiate strains likely to produce known lipopeptides from producers of potentially novel lipopeptides. This work provides a valuable tool that can also be integrated in a genome mining strategy and adapted for the typing of other specialized metabolites.

Downsizing antibodies: Towards complementarity-determining region (CDR)-based peptide mimetics.

Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15-50 kDa), together with smaller synthetic peptides (ca. 1-3 kDa) derived from the antibodies' crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.

Modulation of Arabidopsis root growth by specialized triterpenes.

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.

Do Aptamers Always Bind? The Need for a Multifaceted Analytical Approach When Demonstrating Binding Affinity between Aptamer and Low Molecular Weight Compounds.

In this manuscript, we compare different analytical methodologies to validate or disprove the binding capabilities of aptamer sequences. This was prompted by the lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of independent yet inconsistent data sets in the literature. As an example, we chose three aptamers with a reported affinity in the nanomolar range for ampicillin, a ß-lactam antibiotic, used as biorecognition elements in several detection strategies described in the literature. Application of a well-known colorimetric assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting results with respect to the original report. Therefore, ampicillin binding was evaluated in solution using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and 1H-nuclear magnetic resonance spectroscopy (1H NMR). By coupling the thermodynamic data obtained with ITC with the structural information on the binding event given by native nESI-MS and 1H NMR we could verify that none of the ampicillin aptamers show any specific binding with their intended target. The effect of AuNPs on the binding event was studied by both ITC and 1H NMR, again without providing positive evidence of ampicillin binding. To validate the performance of our analytical approach, we investigated two well-characterized aptamers for cocaine/quinine (MN4), chosen for its nanomolar range affinity, and l-argininamide (1OLD) to show the versatility of our approach. The results clearly indicate the need for a multifaceted analytical approach, to unequivocally establish the actual detection potential and performance of aptamers aimed at small organic molecules.

Cyclic lipopeptide-producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils.

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Tethered imidazole mediated duplex stabilization and its potential for aptamer stabilization.

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pKa. Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pKa-motif responsible for these duplex-stabilizing and pKa-modulating properties. Here, the robustness and utility of this pKa-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pKa-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pKa values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages.

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.

Synthesis and Conformational Properties of 3,4-Difluoro-l-prolines.

Fluorinated proline derivatives have found diverse applications in areas ranging from medicinal chemistry over structural biochemistry to organocatalysis. Depending on the stereochemistry of monofluorination at the proline 3- or 4-position, different effects on the conformational properties of proline (ring pucker, cis/ trans isomerization) are introduced. With fluorination at both 3- and 4-positions, matching or mismatching effects can occur depending on the relative stereochemistry. Here we report, in full, the syntheses and conformational properties of three out of the four possible 3,4-difluoro-l-proline diastereoisomers. The yet unreported conformational properties are described for (3 S,4 S)- and (3 R,4 R)-difluoro-l-proline, which are shown to bias ring pucker and cis/ trans ratios on the same order of magnitude as their respective monofluorinated progenitors, although with significantly faster amide cis/ trans isomerization rates. The reported analogues thus expand the scope of available fluorinated proline analogues as tools to tailor proline's distinct conformational and dynamical properties, allowing for the interrogation of its role in, for instance, protein stability or folding.

The 1,3-diyne linker as a rigid "i,i+7" staple for α-helix stabilization: Stereochemistry at work.

Short alphahelical peptide sequences were stabilized through Glaser-Hay couplings of propargylated l- and/or d-serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d-Ser (i), l-Ser (i+7) combination was applied. In case two l-Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.

Sequence-Specific DNA Binding by Noncovalent Peptide-Azocyclodextrin Dimer Complex as a Suitable Model for Conformational Fuzziness.

Transcription factors are proteins lying at the endpoint of signaling pathways that control the complex process of DNA transcription. Typically, they are structurally disordered in the inactive state, but in response to an external stimulus, like a suitable ligand, they change their conformation, thereby activating DNA transcription in a spatiotemporal fashion. The observed disorder or fuzziness is functionally beneficial because it can add adaptability, versatility, and reversibility to the interaction. In this context, mimetics of the basic region of the GCN4 transcription factor (Tf) and their interaction with dsDNA sequences would be suitable models to explore the concept of conformational fuzziness experimentally. Herein, we present the first example of a system that mimics the DNA sequence-specific recognition by the GCN4 Tf through the formation of a non- covalent tetra-component complex: peptide-azoß-CyD(dimer)-peptide-DNA. The non-covalent complex is constructed on the one hand by a 30 amino acid peptide corresponding to the basic region of GCN4 and functionalized with an adamantane moiety, and on the other hand an allosteric receptor, the azoCyDdimer, that has an azobenzene linker connecting two ß-cyclodextrin units. The azoCyDdimer responds to light stimulus, existing as two photo-states: the first thermodynamically stable with an E:Z isomer ratio of 95:5 and the second obtained after irradiation with ultraviolet light, resulting in a photostationary state with a 60:40 E:Z ratio. Through electrophoretic shift assays and circular dichroism spectroscopy, we demonstrate that the E isomer is responsible for dimerization and recognition. The formation of the non-covalent tetra component complex occurs in the presence of the GCN4 cognate dsDNA sequence ('5-..ATGA cg TCAT..-3') but not with ('5-..ATGA c TCAT..-3') that differs in only one spacing nucleotide. Thus, we demonstrated that the tetra-component complex is formed in a specific manner that depends on the geometry of the ligand, the peptide length, and the ds DNA sequence. We hypothesized that the mechanism of interaction is sequential, and it can be described by the polymorphism model of static fuzziness. We argue that chemically modified peptides of the GCN4 Tf are suitable minimalist experimental models to investigate conformational fuzziness in protein-DNA interactions.

Conformation and Dynamics of the Cyclic Lipopeptide Viscosinamide at the Water-Lipid Interface.

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds.

Pseudomonas sp. COW3 Produces New Bananamide-Type Cyclic Lipopeptides with Antimicrobial Activity against Pythium myriotylum and Pyricularia oryzae.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

Colloidal CdSe Nanoplatelets, A Model for Surface Chemistry/Optoelectronic Property Relations in Semiconductor Nanocrystals.

While the surface termination of quasi-spherical metal chalcogenide nanocrystals or quantum dots has been widely investigated, it remains unclear whether the ensuing surface chemistry models apply to similar nanocrystals with anisotropic shapes. In this work, we report on the surface-chemistry of 2D CdSe nanoplatelets, where we make use of an improved synthesis strategy that yields stable and aggregation free nanoplatelet suspensions with a photoluminescence quantum yield as high as 55%. We confirm that such nanoplatelets are enriched in Cd and, by means of 1H nuclear magnetic resonance spectroscopy, we show that the Cd-rich surface is terminated by X-type carboxylate ligands. Not unlike CdSe quantum dots (QDs), entire cadmium carboxylate entities can be displaced by the addition of amines, and the desorption isotherm points toward a considerable binding site heterogeneity. Moreover, we find that even the slightest displacement of cadmium carboxylate ligands quenches the nanoplatelet photoluminescence. These experimental findings are further confirmed by density functional theory (DFT) calculations on a 5 monolayer model CdSe nanoplatelet. These simulations show that the most labile ligands are located in the vicinity of facet edges, and that the displacement of ligands from such edge sites creates midgap states that can account for the observed photoluminescence quenching. Next to extending surface chemistry insights from colloidal QDs to nanoplatelets, this work indicates that CdSe nanoplatelets constitute a unique nanocrystal model system to establish a comprehensive description of midgap trap states, which includes their structural, chemical, and electronic properties.