Mechanism of penetration of Antp(43-58) into membrane bilayers.

Antp(43-58) is one of many peptides with basic and aromatic residues capable of crossing cell membranes efficiently in a receptor-independent manner. The basic-aromatic motif is responsible for peptide binding to the negatively charged surface of membrane bilayers. However, the mechanism of membrane penetration is unclear. We use high-resolution (1)H solution NMR methods to establish the location of the Antp(43-58) peptide bound to membrane bicelles composed of DMPC, DMPG, and DHPC, and compare it to the location of an Antp(43-58) variant which is not able to cross cell membranes. Two critical tryptophans are substituted with phenylalanine in this variant (W48F and W56F). Additional (31)P and (2)H NMR measurements of membrane bicelles are used to probe the changes in orientation of the lipid headgroups and the changes in the mobility or segmental order of the lipid acyl chains upon peptide binding. We find that Trp48 and Trp56 of Antp(43-58) insert into the hydrophobic core of the membrane and that this induces a change in the orientation of the negatively charged DMPG headgroups. The depth of insertion and the change in lipid orientation are concentration-dependent and argue for an electroporation-like mechanism for membrane penetration.