Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NH2 and its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e. DLeu14 and DSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive α-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.

The fluorescence and infrared absorption probe para-cyanophenylalanine: Effect of labeling on the behavior of different membrane-interacting peptides.

Total syntheses and complete characterizations of singly substituted PheCN -based analogs of alamethicin AlaP, which is active on model and natural membranes, and the TM peptide, which inserts in a transmembrane orientation in lipid bilayers, are reported. The syntheses of the AlaP analogs were performed in solution, while those of TM and its analogs were carried out by solid phase. Using the cyanophenyl fluorescence and infrared (IR) absorption probe, an in-depth investigation of the self-association, membrane-interacting, permeabilizing, and orientation properties of these peptides were conducted. The aromatic residue incorporated induces only a negligible modification to the properties of the parent peptides. The PheCN IR absorption band was located between 2228 and 2230 cm(-1) for all peptides, irrespective of the position of labeling. By contrast, as the width of this band varied significantly with the depth of probe insertion in the bilayer, it could represent a good marker of the PheCN position in phospholipid membranes.

4-Cyano-α-methyl-l-phenylalanine as a spectroscopic marker for the investigation of peptaibiotic-membrane interactions.

Two analogs of the ten-amino acid residue, membrane-active lipopeptaibiotic trichogin GA IV, mono-labeled with 4-cyano-α-methyl-L-phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid-phase methodology and conformationally characterized. The single modification was incorporated either at the N-terminus (position 1) or near the C-terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α-aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT-IR absorption, CD, and 2D-NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide-membrane interactions were assessed by fluorescence and ATR-IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4-cyanobenzyl chromophore are sensitive markers of the local microenvironment.

Aggregation determines the selectivity of membrane-active anticancer and antimicrobial peptides: The case of killerFLIP.

Host defense peptides selectively kill bacterial and cancer cells (including those that are drug-resistant) by perturbing the permeability of their membranes, without being significantly toxic to the host. Coulombic interactions between these cationic and amphipathic peptides and the negatively charged membranes of pathogenic cells contribute to the selective toxicity. However, a positive charge is not sufficient for selectivity, which can be achieved only by a finely tuned balance of electrostatic and hydrophobic driving forces. A common property of amphipathic peptides is the formation of aggregated structures in solution, but the role of this phenomenon in peptide activity and selectivity has received limited attention. Our data on the anticancer peptide killerFLIP demonstrate that aggregation strongly increases peptide selectivity, by reducing the effective peptide hydrophobicity and thus the affinity towards membranes composed of neutral lipids (like the outer layer of healthy eukaryotic cell membranes). Aggregation is therefore a useful tool to modulate the selectivity of membrane active peptides and peptidomimetics.

Structural organization of lipid-functionalized-Au nanoparticles.

Gold nanoparticles (AuNPs) are considered suitable systems for drug delivery and diagnostics with several applications in biomedicine. Size, shape and surface functionalization of these nanoparticles are important parameters influencing their behavior in a biological environment. This study describes the preparation and the characterization of lysophosphocholine coated AuNPs by means of Small Angle Neutron Scattering (SANS), Electron Paramagnetic Resonance (EPR) and Fluorescence Spectroscopy. In particular the structure of the functionalized AuNP suspension, as well as the physical properties, of the nanoparticle organic coating are discussed. The experimental results indicated that functionalized lysophosphocholine-AuNPs form aggregates, which are composed by nanoparticles with core-shell structure. Nevertheless, the nanoparticle suspension resulted to be stable, without significant structural rearrangements even when the temperature was increased to 50 °C. At the same time, experimental evidences also suggested that the 18LPC layer around AuNPs presented a reduced chain packing compared to pure 18LPC aggregates.