Biophysical Characterization of Peptide Membrane Interactions and Membrane Permeabilization

Abstract

Certain membrane-active amphipathic peptides, such as antimicrobial peptides, possess a characteristic feature of having several cationic amino acid residues at either N- or C-terminus. In an attempt to determine the functional significance of C-terminal cationic residues, we have studied the membrane binding mode and membrane pore formation by a 20-amino acid synthetic amphipathic peptide. Two cationic residues were replaced to Leu or Glu to see how the membrane binding and permeabilization activities of the peptide are modulated by charge neutralization and charge reversal mutations. The depth of membrane insertion of the wild type and mutant peptides was determined by measuring the quenching of the fluorescence of a single, C-terminally located tryptophan by dibromo-phosphatidylcholines brominated at various positions of the acyl chains, using pure POPC and 70% POPC + 30% POPG in lipid vesicles. In case of pure POPC membranes, Trp in all three peptides was located at around 9 Å from the membrane center, while for 30% POPG, Trp was inserted much deeper. Studies on peptide-mediated calcein release from phospholipid vesicles were conducted to see if the peptides are able to cause membrane pore formation and content release. Peptides with positively charged residues showed maximum calcein release for both lipid compositions whereas negative or hydrophobic residues were less effective in vesicle permeabilization. Preliminary ATR-FTIR experiments using both POPC and POPG have been performed to identify the orientation of the peptides within the lipid bilayer and their effect of the lipid order.