The amphipathic helix concept: length effects on ideally amphipathic LiKj(i=2j) peptides to acquire optimal hemolytic activity

Abstract

In a minimalist approach to modeling lytic toxins, amphipathic peptides of LiKj with i=2j composition and whose length varies from 5 to 22 residues were studied for their ability to induce hemolysis and lipid vesicle leakage. Their sequences were designed to generate ideally amphipathic alpha helices with a single K residue per putative turn. All the peptides were lytic, their activities varying by more than a factor of 103 from the shortest 5-residue-long peptide (5-mer) to the longest 22-mer. However, there was no monotonous increase versus length. The 15-mer was as active as the 22-mer and even more than melittin which is used as standard. Partition coefficients from the buffer to the membrane increased in relation to length up to 12 residues, then weakly decreased to reach a plateau, while they were expected to increase monotonously with peptide length and hydrophobicity as revealed from HPLC retention times. Fluorescence labeling by a dansyl group at the N-terminus, or by a W near the CO-terminus, show that up to 12 residues, the peptides were essentially monomeric while longer peptides strongly aggregated in the solution. Lipid affinity was then controlled by peptide length and was found to be limited by folding and self-association in buffer. The lytic activity resulted both from lipid affinity, which varied by a factor of 20-fold, and from efficiency in disturbing the membrane when bound, the latter steeply and monotonously increasing with length. The 15-residue-long peptide, KLLKLLLKLLLKLLK, had the optimal size for highest lytic activity. The shallow location of the fluorescent labels in the lipids is further evidence for a model of peptides remaining flat at the interface.

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes

“…Due to the lipid affinity of such peptides, there is an increase of lytic activity with chain length. Moreover, in contrast with earlier studies [26], neither a threshold in length nor a matching between peptide length and membrane thickness was observed [21,24,25]. This strongly supports a mechanism…”

“…This was previously found for their efficacy to induce leakage in lipid vesicles and erythrocytes, and was shown to be due to the selfassociation of such a helices in solution [25]. However, unlike haemolysis the bactericidal activity dropped more severely from 18-to 21-residue length before a slight increase for the 22-residue peptide (Fig.…”

“…Due to the lipid affinity of such peptides, there is an increase of lytic activity with chain length. Moreover, in contrast with earlier studies [26], neither a threshold in length nor a matching between peptide length and membrane thickness was observed [21,24,25]. This strongly supports a mechanism of action based upon the invasion of the outer membrane leaflet by the peptides and their insertion in a flat orientation to form ÔraftsÕ or ÔcarpetsÕ [21,24,25,27,28].…”

“…there is a delicate balance between hydrophobicity and charge repulsion (two antagonistic forces) [14,22,25]. Due to the lipid affinity of such peptides, there is an increase of lytic activity with chain length.…”

“…Our goal was to assess whether or not the rules drawn from the studies with model membranes and erythrocytes [21,25] are also valid for bacteria. Mollicutes were chosen as targets because these bacteria are devoid of both a cell wall and an outer membrane [29] making them sensitive to many natural membrane-active peptides.…”