Kinetic Defects Induced by Melittin in Model Lipid Membranes: A Solution Atomic Force Microscopy Study

Abstract

Quantitative characterization of membrane defects (pores) is important for elucidating molecular basis of many membrane active peptides. We study kinetic defects induced by melittin in vesicular and planar lipid bilayers. Fluorescence spectroscopy measurements indicate that melittin induces time-dependent calcein leakage. Solution atomic force microscopy (AFM) is used to visualize melittin induced membrane defects. After initial equilibration, the most probable defect radius is ~3.8 nm in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) bilayers. Unexpectedly, defects become larger with longer incubation, accompanied by substantial shape transformation. The initial defect radius is ~4.7 nm in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. Addition of 30mol% cholesterol to DOPC bilayers suppresses defect kinetics, although the inhibitory impact is negated by longer incubation. Overall, the kinetic rate of defect development follows DLPC > DOPC > DOPC/cholesterol. Kinetic defects are also observed when anionic lipids are present. Based on the observation that defects can occupy as large as 40% of the bilayer surface, we propose a kinetic defect growth model. We also study the effect of melittin on the phase behavior of DOPC/egg-sphingomyelin/cholesterol bilayers. We find that melittin initially suppresses or eliminates liquid-ordered (Lo) domains; Lo domains gradually emerge and become the dominant species with longer incubation. Defects in phase coexisting bilayers have a most probable radius of ~5 nm, and are exclusively localized in the liquid-disordered (Ld) phase. Our experimental data highlight that melittin induced membrane defects are not static; conversely, spontaneous defect growth is intrinsically associated with membrane permeabilization exerted by melittin.

Modulation of lipid membrane structural and mechanical properties by a peptidomimetic derived from reduced amide scaffold

“…2B) [28, 30, 54]. For 0.1 μM melittin, almost no increase of the fluorescence signal is observed over the 30-min incubation period; the increase of the fluorescence signal becomes fairly significant over ~30 min for 0.2 μM melittin; near full leakage is achieved within a few minutes for melittin ≥0.3 μM.…”

“…To support this statement, we expose a POPC/POPG bilayer to the well-studied antimicrobial peptide melittin, which is known to form transmembrane pores (or defects) [28, 54, 58]. After equilibrating with 0.4 μM melittin for ~30 min, the bilayer is perforated with numerous pore-like defects (Fig.…”

“…It is noteworthy that the maximum depth of the defects is smaller than the bilayer thickness (~3–4 nm) [59–61]. This is mainly related to the geometry and size of the AFM tip (and scanning parameters such as tip speed) [54]. Nevertheless, a comparison between Figs.…”

“…Experimental procedure for solution AFM measurements (23°C) has been reported elsewhere [49, 51-54]. Planar bilayers are formed by the SUV rupture and fusion method [34] in a fluid cell (model MTFML-V2) with a freshly cleaved mica film as the substrate.…”

In Situ Atomic Force Microscopy Studies on Nucleation and Self-Assembly of Biogenic and Bio-Inspired Materials

“…They also observed that adding an N-terminal myc-tag to the htt exon1 fragments can prevent the interaction of htt with the bilayer. The interaction between melittin, a 26-residue amphipathic peptide, and lipid membranes has been studied using in situ AFM by Pan et al [159]. Their results showed that melittin induced defects in lipid bilayers, which can be delayed by introducing cholesterol.…”