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

Antimicrobial peptides which adopt mainly or only beta-sheet structures hav e two or more disulfide bonds stabilizing their structure. The disruption o f the disulfide bonds results in most cases in a large decrease in their an timicrobial activity. In the present study we examined the effect of D-amin o acids incorporation on the structure and function of a cytolytic alpha-he lical peptide which acts on erythrocytes and bacteria. The influence of a s ingle or double D-amino acid replacement in alpha-helical peptides on their structure was reported previously in 50% 2,2,2,trifluoroethanol/water [Kra use et al. (1995) Anal. Chem. 67, 252-258]. Here we used Attenuated Total R eflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy and found tha t the predominant structure of the wild-type peptide is alpha-helix in phos pholipid membranes, whereas the structure of the diastereomer is beta-sheet . However, the linear, beta-sheet diastereomer preserved its cytolytic acti vity on bacteria but not on erythrocytes. Previous studies have shown that the ability of antimicrobial peptides to lyse bacteria but not normal mamma lian cells correlated with their ability to disintegrate preferentially neg atively charged, but not zwitterionic phospholipid membranes. In contrast, the diastereomer described here disrupts zwitterionic and negatively charge d vesicles with similar potencies to those of the hemolytic wild-type pepti de. Interestingly, whereas addition of a positive charge to the N-terminus of the wildtype peptide (which caused a minor effect on its structure) incr eased activity only towards some of the bacteria tested, similar modificati on in the diastereomer increased activity towards all of them. Furthermore, the modified wild-type peptide preserved its potency to destabilize zwitte rionic and negatively charged vesicles, whereas the modified diastereomer h ad a reduced potency on zwitterionic vesicles but increased potency on nega tively charged vesicles. Overall our results suggest that this new class of antimicrobial diastereomeric peptides bind to the membrane in a 'carpet-li ke' manner followed by membrane disruption and breakdown, rather than formi ng a transmembrane pore which interfere with the bacterial potential. These studies also open a way to design new broad-spectrum antibacterial peptide s.