Molecular Resolution Imaging of an Antibiotic Peptide in a Lipid Matrix

Abstract

In this work, we show molecular resolution scanning tunneling microscopy (STM) images of gramicidin, a model antibacterial peptide, inserted into a phospholipid matrix. The resolution of the images is superior to that obtained in previous attempts to image gramicidin in a lipid environment using atomic force microscopy (AFM). This breakthrough has allowed visualization of individual peptide molecules surrounded by lipid molecules. We have observed several important features: the peptide molecules do not aggregate, the peptide molecules adopt a single conformation corresponding to a specific ion channel form, and the lipid molecules adjacent to the peptide molecules are systematically longer than those in the lipid matrix. These results constitute a new approach to obtain structural characteristics of antibiotic peptides in lipid assemblies that is necessary for the understanding of their biological activity.

Direct visualization of the alamethicin pore formed in a planar phospholipid matrix

“…3B) and the mean area of the spot is 0.23 ± 0.1 nm 2 . These dimensions are consistent with the average distance between two lipid chains and cross-sectional area of a lipid chain in closely packed monolayers of phospholipids determined by STM and Xray diffraction (27,35,36). They suggest that the contrast in Fig.…”

“…Recently, we obtained molecular resolution images of a gramicidin, a hydrophobic channel forming peptide, dispersed in a monolayer of DMPC (27). The goal of the present study was to use similar methodology to provide complementary information about the structure of the pore formed by the amphiphatic peptide such as alamethicin.…”

Single-molecule studies on individual peptides and peptide assemblies on surfaces

“…The STM images of gD/lipid system show triangular pores corresponding to the molecular structures of gD from the protein data bank [19]. The latter study also proved that the peptide affects only the properties of the lipids next to it, while not the ordering of the lipids in the bulk matrix [19]. These investigations provided direct evidence for the peptide aggregation and peptide-lipid interactions, which is beneficial for the understanding of their biological activities.…”

Can gramicidin ion channel affect the dipole potential of neighboring phospholipid headgroups?

“…Gramicidin has a low solubility in water (≤ 1 × 10 −4 μM according to Hladky and Haydon [32]) and has been found to partition strongly into the hydrophobic region of lipid membranes [33]. Hence, at the 0.1 μM gramicidin concentration adopted herein, much higher than its solubility value in pure water, it is quite reasonable for its concentration in the lipid bilayer to assume a value close to the 1:10 peptide/lipid molar ratio adopted in a theoretical analysis of hydrophobic matching [34], in a densitometry and sound velocimetry study on the effect of gramicidin [35], and in a STM imaging of gramicidin in a dimyristoylphosphatidylcholine (DMPC) monolayer on Au [36]. In particular, the counting of the lipid molecules surrounding each gramicidin channel yields an average number of 7 ± 1 [36], which is consistent with molecular dynamics calculations that assume eight nearestneighbor lipids [37].…”