Bilayer Interactions of Indolicidin, a Small Antimicrobial Peptide Rich in Tryptophan, Proline, and Basic Amino Acids

Abstract

Tryptophan, proline, and basic amino acids have all been implicated as being important in the assembly and structure of membrane proteins. Indolicidin, an antimicrobial 13-residue peptide-amide isolated from the cytoplasmic granules of bovine neutrophils, is highly enriched in these amino acids: five tryptophans, three prolines, three basic residues, and no acidic residues. Consistent with the likely importance of these 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 indolicidin with large unilamellar vesicles (LUV) formed from palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylglycerol (POPG), in order to use it as a model system for studying membrane protein insertion and for evaluating the relative contributions of hydrophobic and electrostatic forces in peptide-bilayer interactions. Equilibrium dialysis measurements indicate that indolicidin binds strongly, 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 necessitated the development of a new equilibrium dialysis method that is described in detail. Tryptophan fluorescence measurements show that indolicidin is located in the bilayer interface and that indole fluorescence is affected by the type of lipid used to form the LUVs. Circular dichroism (CD) measurements reveal unordered conformations in aqueous and bulk organic solutions and a somewhat more ordered, but not alpha-helical, conformation in SDS micelles and lipid bilayers. Fluorescence requenching measurements (Ladokhin et al. 1995. Biophys. J. 69:1964-1971) on vesicles loaded with the fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX), show that indolicidin induces membrane permeabilization. For anionic POPG, leakage is graded with a high preference for the release of cationic DPX over anionic ANTS. For neutral POPC vesicles no such preference is observed. Leakage induction is more effective with POPG vesicles than with POPC vesicles, as judged by three quantitative measures that are developed in the Appendix.

Cation−π Interactions Stabilize the Structure of the Antimicrobial Peptide Indolicidin near Membranes: Molecular Dynamics Simulations

“…(2) Although the average rmsf of the peptide backbone was only slightly higher for W11A than for IND, the average rmsf of residues 8-13 is significantly higher while the average rmsf of residues 1-5 is slightly lower in W11A than in IND. These data indicate that loss of the boat-shaped structure of W11A in DPC is driven by a disordering of residues [8][9][10][11][12][13]. (3) The orientation of the tryptophan side chains had a greater average fluctuation in W11A than for indolicidin in DPC.…”

Length Effects in Antimicrobial Peptides of the (RW)nSeries

“…2). The negative band around 200 nm is characteristic of small unfolded peptides, while the band at 225 nm is due to the indole side chain of W (21,57), as seen in the CD spectrum of a peptide such as GGWGG containing a single chiral W residue (44). Each of the five peptides exhibits distinctive CD spectra in the presence of POPG and POPC.…”

“…CD spectroscopy is widely used to analyze the secondary structure of proteins and peptides because it is extremely sensitive to conformational changes. (RW) n in buffer appears to be unfolded due to the negative band around 200 nm that is characteristic of unfolded model peptides (44); the positive band around 225 nm due to the indole side chain is Trp specific (21,57). Peptide interactions with liposomes are accompanied by distinct CD spectral changes.…”

A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia ( Dahlia merckii )

“…Sphingolipids have not been identified so far as possible targets for antimicrobial peptides. Most antimicrobial peptides studied thus far are known to interact with ubiquitous phosphoglycerolipids (28)(29)(30)(31)(32), explaining their relatively broad antimicrobial spectrum. Nisin, a narrow-spectrum antibacterial peptide produced by some lactic acid bacteria, has been shown to interact specifically with the membrane-bound peptidoglycan precursor lipid II.…”