Interaction of Bee Venom Melittin with Zwitterionic and Negatively Charged Phospholipid Bilayers

Abstract

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

Melittin: a Membrane-active Peptide with Diverse Functions

“…Melittin is known to interact selectively with negatively charged lipids (Beschiaschvili and Seelig 1990;Gromova et al 1992;Kleinschmidt et al 1997;Ghosh et al 1997;Sheynis et al 2003). The affinity of melittin for membranes composed of negatively charged lipids has been shown to be 100-fold greater than for zwitterionic lipids (Batenburg et al 1988;Lee et al 2001).…”

Synthesis of poly(sulfobetaine methacrylate)-grafted chitosan under γ-ray irradiation for alamethicin assembly

“…Spin-labeled lipid probes have been extensively used as a tool for probing the organization and local motion within lipid membranes [46]. In this study, ESR was used to investigate the association of the peptides with the micelles with spin-labeled probes.…”

Effect of micellar charge on the conformation and dynamics of melittin

“…Melittin is known to interact selectively with negatively charged lipids (Beschiaschvili and Seelig 1990;Ghosh et al 1997;Kleinschmidt et al 1997). The affinity of melittin for membranes composed of negatively charged lipids has been shown to be 100-fold greater than for zwitterionic lipids (Batenburg et al 1988;Lee et al 2001).…”