A comparative study on the structure and function of a cytolytic α-helical peptide and its antimicrobial β-sheet diastereomer

Antimicrobial peptides which adopt mainly or only beta-sheet structures have two or more disulfide bonds stabilizing their structure. The disruption of the disulfide bonds results in most cases in a large decrease in their antimicrobial activity. In the present study we examined the effect of D-amino acids incorporation on the structure and function of a cytolytic alpha-helical peptide which acts on erythrocytes and bacteria. The influence of a single or double D-amino acid replacement in alpha-helical peptides on their structure was reported previously in 50% 2,2,2,trifluoroethanol/water [Krause et al. (1995) Anal. Chem. 67, 252-258]. Here we used Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy and found that the predominant structure of the wild-type peptide is alpha-helix in phospholipid membranes, whereas the structure of the diastereomer is beta-sheet. However, the linear, beta-sheet diastereomer preserved its cytolytic activity on bacteria but not on erythrocytes. Previous studies have shown that the ability of antimicrobial peptides to lyse bacteria but not normal mammalian cells correlated with their ability to disintegrate preferentially negatively charged, but not zwitterionic phospholipid membranes. In contrast, the diastereomer described here disrupts zwitterionic and negatively charged vesicles with similar potencies to those of the hemolytic wild-type peptide. Interestingly, whereas addition of a positive charge to the N-terminus of the wildtype peptide (which caused a minor effect on its structure) increased activity only towards some of the bacteria tested, similar modification in the diastereomer increased activity towards all of them. Furthermore, the modified wild-type peptide preserved its potency to destabilize zwitterionic and negatively charged vesicles, whereas the modified diastereomer had a reduced potency on zwitterionic vesicles but increased potency on negatively charged vesicles. Overall our results suggest that this new class of antimicrobial diastereomeric peptides bind to the membrane in a 'carpet-like' manner followed by membrane disruption and breakdown, rather than forming a transmembrane pore which interfere with the bacterial potential. These studies also open a way to design new broad-spectrum antibacterial peptides.