Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the ␤-stranded gramicidin S and the ␣-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.

We propose a concept for the folding and self-assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other membrane proteins based on electrostatic "charge zippers." Each subunit of TatA consists of a transmembrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complementary to the charge pattern on the APH, suggesting that the protein can be "zipped up" by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a transmembrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by monitoring the monomer-oligomer equilibrium of specific charge mutants. Similar "charge zippers" are proposed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral E(RNS) protein.

Many vital cellular processes, such as protein translocation, proton transport or\ud molecular recognition, are mediated by self assembling membrane proteins.\ud We have investigated the Twin-arginine translocase (TatA) complex, which\ud forms transient pores through which proteins are translocated through the\ud membrane. We postulated that complex formation is electrostatically driven\ud by formation of salt bridges between amphiphilic transmembrane segments\ud of the individual monomers and developed a structure-based model for this\ud process[1].\ud We studied the formation of oligomers of different sizes by structure-based[2]\ud MD simulations in combination with NMR constraints and a hydrophobic-slab\ud implicit membrane model. Starting from isolated monomers, distributed far\ud apart from each other, we observed the formation of stable TatA oligomers\ud on the basis of the postulated interactions. The dimensions of the resulting\ud TatA complex agreed well with experimental electron microscopy measure-\ud ments[3] and the postulated interactions were confirmed by subsequent muta-\ud tion studie