Abstract
Hundreds of cationic antimicrobial and cell-penetrating peptides (CPPs) form amphipathic α-helices when bound to lipid membranes. Here, we test two hypotheses for the differences in the ability of these peptides to translocate across membranes. The first, which we now call the hydrophobicity hypothesis, is that peptide translocation is determined by the Gibbs energy of insertion into the bilayer from the membrane interface. The second, which we call the charge-distribution hypothesis, is that translocation is determined by whether the distribution of cationic residues in the peptide can transiently stabilize a high-energy inserted intermediate by forming salt bridges to the phosphates of lipid headgroups. To test these hypotheses, we measured translocation of two series of peptide variants. The first series was based on TP10W, a peptide derived from the amphipathic CPP transportan 10; the second was based on DL1a, a synthetic peptide derived from staphylococcal δ-lysin. The peptides in those two series had small sequence changes relative to TP10W and DL1a: either single-residue substitutions or two-residue switches, which were designed to increase or decrease translocation differently according to the two hypotheses. We found that with regard to the changes introduced in the sequences, five out of six peptide variants translocated in agreement with the charge-distribution hypothesis, whereas none showed agreement with the hydrophobicity hypothesis. We conclude that large effects on translocation are probably determined by hydrophobicity, but the fine tuning appears to arise from the distribution of cationic residues along the peptide sequence.