title: Gramicidin A disassembles large conductive clusters of its lysine-substituted derivatives in lipid membranes.
abstract: N-terminally substituted lysine derivatives of gramicidin A (gA), [Lys1]gA and [Lys3]gA, but not glutamate- or aspartate-substituted peptides have been previously shown to cause the leakage of carboxyfluorescein from liposomes. Here, the leakage induction was also observed for [Arg1]gA and [Arg3]gA, while [His1]gA and [His3]gA were inactive at neutral pH. The Lys3-containing analogue with all tryptophans replaced by isoleucines did not induce liposome leakage, similar to gA. This suggests that the presence of both tryptophans and N-terminal cationic residues is critical for pore formation. Remarkably, the addition of gA blocked the leakage induced by [Lys3]gA. By examining with fluorescence correlation spectroscopy the peptide-induced leakage of fluorescent markers from liposomes, we estimated the diameter of pores responsible for the leakage to be about 1.6 nm. Transmission electron cryo-microscopy imaging of liposomes with [Lys3]gA showed that the liposomal membranes contained high electron density particles with a size of about 40 Å, suggesting the formation of peptide clusters. No such clusterization was observed in liposomes incorporating gA or a mixture of gA with [Lys3]gA. Three-dimensional reconstruction of the clusters was compatible with their pentameric arrangement. Based on experimental data and computational modeling, we suggest that the large pore formed by [Lys3]gA represents a barrel-stave oligomeric cluster formed by antiparallel double-stranded helical dimers (DH). In a tentative model, the pentamer of dimers may be stabilized by aromatic Trp-Trp and cation-π Trp-Lys interactions between the neighboring DHs. The inhibiting effect of gA on the [Lys3]gA-induced leakage can be attributed to breaking of cation-π interactions, which prevents peptide clusterization and pore formation.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia. antonen@genebee.msu.ru.
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Antonenko
authors.forename: Grigory S
authors.initials: GS
authors.lastname: Gluhov
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Firsov
authors.forename: Irina D
authors.initials: ID
authors.lastname: Pogozheva
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Kovalchuk
authors.forename: Evgeniya V
authors.initials: EV
authors.lastname: Pechnikova
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kotova
authors.forename: Olga S
authors.initials: OS
authors.lastname: Sokolova
mesh_terms: D006096:Gramicidin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry*; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D013050:Spectrometry, Fluorescence
chemical_list: D008081:Liposomes; D006096:Gramicidin; D008239:Lysine