Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides

To investigate the influence of proline residues on the activity of ct-heli cal peptides, variants were synthesized with insertions of proline residues to create peptides without proline, or with one or two prolines; The influ ence of the proline-induced bends was assessed by circular dichroism in the presence of liposomes, and the ability of the peptides to kill microorgani sms, to permeabilize the outer and cytoplasmic membranes of Escherichia col i, to bind to liposomes, to form channels in planar lipid bilayers, and to synergize with conventional antibiotics. Representative peptides adopted al pha-helical conformations in phosphatidylcholine/phosphatidylglycerol (POPC /POPG, 7:3) liposomes as well as in 60% trifluoro-ethanol solution, as reve aled by circular dichroism (CD) spectroscopy, However, the percent of helic ity decreased as the number of proline residues increased. Tryptophan fluor escence spectroscopy showed that all of these peptides inserted into the me mbranes of liposomes as indicated by a blue shift in the emission maximum a nd an increase in the fluorescence intensity of the single tryptophan at re sidue 2. Quenching experiments further prove that the tryptophan residue wa s no longer accessible to the aqueous quencher KI. The peptide that lacked proline exhibited the highest activity [minimal inhibitory concentrations ( MICs) of 0.5-4 mu g/mL] against all tested Gram-negative and Gram-positive bacteria, but was hemolytic at 8 mu g/mL. The single-proline peptides exhib ited intermediate antibacterial activity. Peptides with two proline residue s were even less active with moderate MICs only against E. coli. With only one exception from each group, the peptides were nonhemolytic. The ability of the peptides to demonstrate synergy in combination with conventional ant ibiotics increased as the antibacterial effectiveness decreased. All peptid es bound to bacterial lipopolysaccharide and permeabilized the outer membra ne of E. coli to similar extents. However, their ability to permeabilize th e cytoplasmic membrane of E. coli as assessed by the unmasking of cytoplasm ic beta-galactosidase decreased substantially as the number of proline resi dues increased. Correspondingly, increasing the number of proline residues caused a decreased ability to form channels in planar Lipid bilayers, and t he hemolytic, proline-free peptide tended to cause rapid breakage of planar membranes. Thus, the number of bends created by insertion of proline resid ues is an important determinant of antimicrobial, hemolytic, and synergisti c activity.