Sara Bobone

Membrane thickness and the mechanism of action of the short peptaibol trichogin GA IV

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug‐resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α‐helical AMPs surprisingly have a helix‐breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane‐active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright

The thin line between cell-penetrating and antimicrobial peptides: the case of Pep-1 and Pep-1-K ‡

Cell‐penetrating peptides (CPPs) are cationic oligopeptides able to translocate across biological membranes without perturbing them, while antimicrobial peptides (AMPs) kill bacteria mainly by disrupting their membranes. The two peptide classes share several characteristics (charge, amphipathicity, helicity, and length), and therefore the molecular properties discriminating between the two different bioactivities are not clear. Pep‐1‐K (KKTWWKTWWTKWSQPKKKRKV) is a new AMP derived from the widely studied CPP Pep‐1 (KETWWETWWTEWSQPKKKRKV), or ‘Chariot’, known for its ability to carry large cargoes across biological membranes. Pep‐1‐K was obtained from Pep‐1 by substituting the three Glu residues with Lys, to increase its cationic character. Previous studies showed that these modifications endow Pep‐1‐K with a potent antimicrobial activity, with MICs in the low micromolar range. Here, we characterized the interaction of Pep‐1 and Pep‐1‐K with model membranes to understand the reason for the antimicrobial activity of Pep‐1‐K. The data show that this peptide causes vesicle aggregation, perturbs membrane order, and induces the leakage of ions, but not of larger solutes, while these effects were not observed for Pep‐1. These differences are likely due, at least in part, to the higher affinity of Pep‐1‐K toward anionic bilayers, which mimick the composition of bacterial membranes. Copyright

The lipid dependence of antimicrobial peptide activity is an unreliable experimental test for different pore models.

Antimicrobial peptides usually kill bacteria by making their membranes permeable. Two main models (barrel-stave and Shai-Matsuzaki-Huang) have been proposed to describe the peptide-induced pores. Although several experimental tests can be exploited to discriminate between these two models, the dependence of peptide activity on lipid properties (intrinsic curvature and membrane thickness) is routinely used for this purpose. Here, we show that, contrary to what is currently accepted, this criterion is unreliable.

On the purported “backbone fluorescence” in protein three-dimensional fluorescence spectra

In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220–230 nm. In the last 10 years we have observed a worrying increase in the number of articles claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220–230 nm is due to the excitation of Tyr and/or Trp, with subsequent emission from the lowest excited state (i.e. the same as obtained with 280 nm excitation) in agreement with Kashas rule. Therefore, this fluorescence peak does not provide any information on backbone conformation, but simply reports on the local environment around the aromatic side chains, just as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim.

Liposome-Templated Hydrogel Nanoparticles as Vehicles for Enzyme-Based Therapies

Several diseases are related to the lack or to the defective activity of a particular enzyme; therefore, these proteins potentially represent a very interesting class of therapeutics. However, their application is hampered by their rapid degradation and immunogenic side effects. Most attempts to increase the bioavailability of therapeutic enzymes are based on formulations in which the protein is entrapped within a scaffold structure but needs to be released to exert its activity. In this work, an alternative method will be described, designed to keep the enzyme in its active form inside a nanoparticle (NP) without the need to release it, thus maintaining the protective action of the nanoscaffold during the entire period of administration. In this approach, liposomes were used as nanotemplates for the synthesis of polyacrylamide hydrogel NPs under nondenaturing conditions, optimizing the polymer properties to obtain a mesh size small enough to limit the enzyme release while allowing the free diffusion of its substrates and products. The enzyme Cu, Zn-superoxide dismutase was chosen as a test case for this study, but our results indicate that the approach is generalizable to other enzymes. Biocompatible, size-tunable nanoparticles have been obtained, with a good encapsulation efficiency (37%), in which the enzyme maintains its activity. This system represents a promising tool for enzyme-based therapy, which would protect the protein from antibodies and degradation while allowing it to exert its catalytic activity.

4-Cyano-α-methyl-l-phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane 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.

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.

From liposomes to cells: Filling the gap between physicochemical and microbiological studies of the activity and selectivity of host-defense peptides

Host‐defense peptides (HPDs) are bactericidal and immunomodulatory molecules, part of the innate immune system of many organisms, including man. They kill bacteria mostly by perturbing their membranes, and for this reason they are a promising class of molecules to fight drug‐resistant microbes. However, their success towards clinical application is still limited, partly due to many unanswered questions on their activity and function. Our current understanding of HDPs has been reached by two parallel, but largely independent, approaches: microbiological studies on HDP effects on cells, and physicochemical investigations on model membranes. All current models for the mechanisms of HDP membrane perturbation and cell selectivity were derived from the latter kind of studies, but their relevance for real cells still had to be demonstrated. In the last few years, several studies led to quantitative insights into HDP behavior directly in cells: membrane‐binding and peptide‐induced pores in bacteria and liposomes were compared; the number of cell‐bound peptide molecules needed to kill a bacterium was determined; the variation of peptide activity and toxicity with the density of cells was characterized; selectivity was examined in a mixture of target and host cells; the sequence of events leading to bacterial death was observed in real time by microscopy on single cells. Overall, these approaches led to a new understanding of HDPs that will be helpful for their development into effective antibiotic drugs.