Pore Formation Mechanisms of Melittin-Like Membrane-Active Peptides

Abstract

Pore formation by membrane-active peptides is believed to play a key role in the mechanism of action of antimicrobial peptides. However, at present the process of spontaneous pore formation, the pore structure, and the pore lifetime in the bilayer remain unknown. This is largely due to experimental difficulties in resolving transient protein structures and dynamics in fluid-phase lipid bilayers. Here we employ a mixture of unbiased multi-microsecond molecular dynamics simulations and experimental techniques to study the process of pore formation and conductance of melittin, a pore-forming membrane active peptide from honey-bee venom, as well as synthetically evolved gain-of-function variants of melittin, which have recently been developed by William Wimley at Tulane University.Using spectroscopic techniques we show that gain-of-function variants are able to leak larger dyes from unilamellar vesicles than melittin. Like melittin gain-of-function variants result in virtually complete leakage of the dye from vesicles. However, the concentration of peptide required to achieve leakage is significantly lower for gain-of-function variants than for melittin. This suggests that these peptides form larger pores than melittin and that these pores are much more stable, remaining functional over the lifetime of the leakage experiment. Simulations of both melittin and gain-of-function variants reveal a wealth of atomic detail information about transient processes such as peptide absorption, folding, and oligomeric assembly, as well as the equilibrium structural ensemble and stability, which were verified using circular dichroism, fluorescence, and electrochemical impedance spectroscopy.