7 research outputs found
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 pO<sub>2</sub>
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 O<sub>2</sub> 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 pO<sub>2</sub>. 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 O<sub>2</sub>. 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 O<sub>2</sub>, 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