title: 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 the 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 30 mol % 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; and 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.
authors.affiliation: Department of Physics, University of South Florida , Tampa, Florida 33620, United States.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Pan
authors.affiliation: Department of Physics, University of South Florida , Tampa, Florida 33620, United States.
authors.forename: Nawal K
authors.initials: NK
authors.lastname: Khadka
mesh_terms: D002784:Cholesterol / Q000737:chemistry; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008555:Melitten / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D010713:Phosphatidylcholines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D008051:Lipid Bilayers; D010713:Phosphatidylcholines; C015153:1,2-dilauroylphosphatidylcholine; D008555:Melitten; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine