Novel Antimicrobial Peptides from the Arctic Polychaeta Nicomache minor Provide New Molecular Insight into Biological Role of the BRICHOS Domain.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.

Breaking the carboxyl rule: lysine 96 facilitates reprotonation of the Schiff base in the photocycle of a retinal protein from Exiguobacterium sibiricum.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

The pH-dependent induction of lipid membrane ionic permeability by N-terminally lysine-substituted analogs of gramicidin A.

Insertion of charged groups at the N-terminus of the gramicidin A (gA) amino acid sequence is considered to be fatal for peptide channel-forming activity because of hindrance to the head-to-head dimer formation. Here the induction of ionic conductivity in planar bilayer lipid membranes (BLM) was studied with gA analogs having lysine either in the first ([Lys1]gA) or the third ([Lys3]gA) position. If added to the bathing solution at neutral or acidic pH, these analogs, being protonated and thus positively charged, were unable to induce ionic current across BLM. By contrast, at pH 11 the induction of BLM conductivity was observed with both lysine-substituted analogs. Based on the dependence of the macroscopic current on the side of the peptide addition, sensitivity to calcium ions and susceptibility to sensitized photoinactivation, as well as on the single-channel properties of the analogs, we surmise that at alkaline pH [Lys1]gA formed channels with predominantly single-stranded structure of head-to-head helical dimers, whereas [Lys3]gA open channels had the double-stranded helical structure. CD spectra of the lysine-substituted analogs in liposomes were shown to be pH-dependent.

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A.

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.