BILAYER INTERACTIONS OF INDOLICIDIN, A SMALL ANTIMICROBIAL PEPTIDE RICH IN TRYPTOPHAN, PROLINE, AND BASIC-AMINO-ACIDS

Tryptophan, proline, and basic amino acids have all been implicated as being important in the assembly and structure of membrane proteins. I ndolicidin, an antimicrobial 13-residue peptide-amide isolated from th e cytoplasmic granules of bovine neutrophils, is highly enriched in th ese amino acids: five tryptophans, three prolines, three basic residue s, and no acidic residues. Consistent with the likely importance of th ese amino acids in membrane protein assembly, indolicidin is known to be highly membrane-active and is believed to act by disruption of cell membranes. We have, therefore, examined the interactions of native in dolicidin with large unilamellar Vesicles (LUV) formed from palmitoylo leoytphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylglycer ol (POPG), in order to use it as a model system for studying membrane protein insertion and for evaluating the relative contributions of hyd rophobic and electrostatic forces in peptide-bilayer interactions. Equ ilibrium dialysis measurements indicate that indolicidin binds strongl y, but reversibly, to both neutral POPC and anionic POPG vesicles with free energies of transfer of -8.8 +/- 0.2 and -11.5 +/- 0.4 kcal/mol, respectively. The extremely strong partitioning into POPG vesicles ne cessitated the development of a new equilibrium dialysis method that i s described in detail. Tryptophan fluorescence measurements show that indolicidin is located in the bilayer interface and that indole fluore scence is affected by the type of lipid used to form the LUVs. Circula r dichroism (CD) measurements reveal unordered conformations in aqueou s and bulk organic solutions and a somewhat more ordered, but not alph a-helical, conformation in SDS micelles and lipid bilayers. Fluorescen ce requenching measurements (Ladokhin et al. 1995. Biophys. J. 69:1964 -1971) on vesicles loaded with the fluorophore/quencher pair 8-aminona pthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX), show that indolicidin induces membrane permeabilization. For a nionic POPG, leakage is graded with a high preference for the release of cationic DPX over anionic ANTS. For neutral POPC vesicles no such p reference is observed. Leakage induction is more effective with POPG v esicles than with POPC vesicles, as judged by three quantitative measu res that are developed in the Appendix.