Membrane Insertion and Bilayer Perturbation by Antimicrobial Peptide CM15

Abstract

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2-9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an alpha-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2-3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup

“…Unordered peptides and proteins show a strong negative peak centered in the 195-200 nm region and substantially weaker, positive or negative signals above 215 nm. Taking these into consideration, the CD curve of CM15 measured in water (pH ~ 6) is characteristic to the dominant contribution of the unordered or random coil structure (FIGURE 1) (Pistolesi et al 2007). In full concordance with this, quantitative analysis of the CD data yielded 62% random coil and 26% β-sheet with no α-helical content.…”

Novel antimicrobial peptides that exhibit activity against select agents and other drug resistant bacteria

“…required for antibiotic activity. 18,[26][27][28] The 'threshold crossing' concentration is very different than the solution concentration of the peptide, it is the concentration required on the surface to disrupt the membrane. 18,[26][27][28] Melo et al report, 'the local peptide concentration in a model bacterial membrane is approximately 10,000 times higher than its concentration in the aqueous phase'.…”

“…18,[26][27][28] The 'threshold crossing' concentration is very different than the solution concentration of the peptide, it is the concentration required on the surface to disrupt the membrane. 18,[26][27][28] Melo et al report, 'the local peptide concentration in a model bacterial membrane is approximately 10,000 times higher than its concentration in the aqueous phase'. 18 The observed MIC values for a particular AMP is the measurable macroscopic threshold indicating the onset of antibiotic activity, thus the relationship between a low lipid to peptide ratio and the MIC values clearly indicate that a high membrane coverage by the peptide may be a requirement for antibiotic activity.…”

Lysine-Enriched Cecropin-Mellitin Antimicrobial Peptides with Enhanced Selectivity

“…The results of previous studies have shown that CM15 is ␣-helical in the presence of helix-promoting solvents (15) or membranes (15,16). In an ␣-helical configuration, CM15 has an almost-ideal amphipathic distribution of amino acid side chains (3,16).…”