CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix

Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the c ytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al, [(1996) J, Biol, Chem. 271, 19298] suggested tha t indolicidin forms a poly-L-proline II helix based upon the circular dichr oism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in t he CD spectra of native indolicidin in various solvents or when bound to mi celles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794], We inter preted the spectra as arising from unordered and/or beta-turn structures, b ut noted a sharp negative band at 227 nm arising from the tryptophan residu es that would mask spectral features characteristic of poly-L-proline II he lix. We have reexamined this issue by means of CD measurements of native in dolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied, To eliminate artifacts associated with tryptophan , we synthesized indolicidin-L and indolicidin-F in which all five tryptoph ans were replaced with leucines or phenylalanines, respectively. The change s in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of p-turns. Fo r the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a po sitive one at 217 nm. This phenomenon, which is absent in indolicidin-L var iants with single Leu-Trp substitutions, is consistent with exciton splitti ng produced by the stacking of indole rings. Type VI turns in model peptide s in aqueous solution are known to be promoted by stacking interactions bet ween cis-proline and neighboring aromatic residues [Yao et al, (1994) J, Mo l. Biol, 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pr o(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn confor mation and revealed the possibility of an accompanying amphipathic structur e. We therefore suggest that turn conformations are the principal structura l motif of indolicidin and that these turns greatly enhance membrane activi ty.