SELF-ASSEMBLY OF DESIGNED ANTIMICROBIAL PEPTIDES IN SOLUTION AND MICELLES

Hydrophobic interactions are responsible for stabilizing leucine zippe rs in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly o f coiled-coils were examined in minimalist antimicrobial peptides desi gned to form amphipathic alpha-helices, The secondary structure of the se peptides was monitored in solution and in diphosphocholine (DPC) mi celles using circular dichroism spectroscopy. The leucine peptides (KL AKLAK)(3) and (KLAKKLA)(n) (n = 3, 4) become alpha-helical with increa sing concentrations of salt, peptide, and DPC. The aggregation state a nd equilibrium constant for self-association of the peptides were meas ured by sedimentation equilibrium. The glycine peptide (KLGKKLG)(3) do es not self-associate, The leucine peptides and phenylalanine peptides (KFAKFAK)(3) and (KFAKKFA)(n) (n = 3, 4) are in a monomer-tetramer eq uilibrium in solution, with the phenylalanine zippers being 2-4 kcal/m ol less stable than the equivalent leucine zippers, Thermodynamic para meters for the association reaction were calculated from the temperatu re dependence of the association constants. Leucine zipper formation h as Delta C-p = 0, whereas phenylalanine zipper formation has a small n egative Delta C-p, presumably due to the removal of the larger surface area of phenylalanine from water. Self-association of tile peptides i s coupled to formation of a hydrophobic core as detected using 1-anili no-naphthalene-8-sulfonate fluorescence. Carboxyfluorescein-labeled pe ptides were used to determine the aggregation stale of (KLAKKLA)(3) an d (KLGKKLG)(3) in DPC micelles, (KLAKKLA)(3) forms dimers, and (KLGKKL G)(3) is a monomer. Aggregation appears to correlate with tile cytotox icity of these peptides.