Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1–18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1–18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1–18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1–18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP

The N‑Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms

Structure–Function Paradigm in Human Myoglobin: How a Single-Residue Substitution Affects NO Reactivity at Low pO₂

This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin’s oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O₂ levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO₂. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O₂. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix–loop–D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O₂, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin–NO formation was experimentally observed through EPR

Circular Dichroism analysis.

<p><b>(A)</b> Conventional CD spectra of both SB056-lin and β-SB056-lin in the presence of POPC/POPG (1/1 mol/mol) SUVs. <b>(B)</b> SRCD spectra of both SB056-lin and β-SB056-lin in the presence of POPC/POPG (1/1 mol/mol) SUVs. SRCD spectra of <b>(C)</b> SB056-lin and <b>(D)</b> β-SB056-lin in the presence of differently charged SUVs.</p

Summary of peptide-lipid interactions.

<p>The proportion of anionic lipids in the vesicles is increased from top to bottom. The behavior of the original SB056-lin peptide is represented on the left hand side, and the sequence-optimized β-SB056-lin on the right. Black arrows of different length and thickness are used to indicate the different binding equilibria. SB056-lin binds only to anionic bilayers, and in a not so well-ordered β-stranded conformation. The sequence optimized β-SB056-lin, on the other hand, forms regular β-strands that self-assemble into extended β-sheets when the negative charge of the bilayer exceeds electro-neutrality of the peptide-lipid system.</p

Schematic representation of the peptides.

<p><b>(A)</b> Original SB056-lin, and <b>(B)</b> sequence-optimized β-SB056-lin. Yellow circles indicate hydrophobic residues, blue ones positively charged amino acids, and cyan indicates the polar serine residue.</p

MIC values of SB056-lin and β-SB056-lin against two Gram-negative and two Gram-positive bacterial strains.

<p>MIC values of SB056-lin and β-SB056-lin against two Gram-negative and two Gram-positive bacterial strains.</p