# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities  
### Antimicrobial Activity  
Once bound to the target membrane, HDPs disrupts the membrane structural integrity and barrier function via a wide range of mechanisms such as pore formation (e.g., barrel stave and toroidal pore) and detergent-like action (e.g., carpet mechanisms), which have already been extensively reviewed [55; 56]. Diastereomers and their all-L counterparts have similar membrane destabilization mechanisms. However, the dependence on either membrane perforation or the detergent effect for membrane destabilization is considerably different for the two classes of peptides. As explained above, diastereomers were found to bind only the surface of simulated mammalian membranes and could not form pores on these membranes. Although the number of peptides tested in the study of [PERSON] and [PERSON] [49] was limited and involved only measurements of model membranes, however it was later shown that the results of this study was indeed general. Circular dichroism analysis performed in a study by [PERSON] and [PERSON] [57] demonstrated that the incorporation of d-AAs was highly disruptive to \(\alpha\)-helical structures and drastically reduced the helical content of diastereomeric peptides. The absence of significant helical structures prevented the diastereomers from forming transmembrane pores due to the lack of a well-structured hydrophobic face [57; 58; 59]. Compared to all-l peptides, the diastereomers that were tested on live bacteria had substantially less hemolytic activity, which was notably correlated with the degree of helicity loss, but retained their antimicrobial activity. Scanning electron microscopy confirmed that the membrane lysis was highly significant, thereby proving that the diastereomers had to function via membrane destabilization, despite their inability to form pores. The authors postulated but did not prove that the carpet mechanism was the mechanism of action. The study of [PERSON] and [PERSON] [57] also indicated that the helical structure was important for the cytotoxicity towards mammalian cells but was not a prerequisite for the lytic activity against bacterial membranes. This finding agrees well with the results of [PERSON] and [PERSON] [49]. Similar conclusions were obtained by many related studies [50; 51; 60; 61; 62], which had demonstrated via tryptophan quenching that diastereomers rely on the carpet mechanism for membrane destabilization. In this process, a large number of peptide molecules bind and envelope the membrane like a carpet and then dissolves the membrane like a detergent. These studies all demonstrated that the incorporation of d-AAs into peptides destabilizes helical structures. The resulting diastereomers had greatly reduced or even abolished hemolytic and cytotoxic effects against mammalian cells but retained their antimicrobial activity. This result was also applicable to venom peptides, such as melittin [61], which is strongly hemolytic. Additionally, [PERSON] and [PERSON] [50] suggested that a specific sequence, length, and position of d-AAs were not a prerequisite for antimicrobial activity. Furthermore, a proper combination of hydrophobicity and positive charge may be sufficient for the design of potent antimicrobial diastereomer HDPs. There are, however, some notable contradictions to this view. [PERSON] et al. [14] have shown that the incorporation of a single d-AA into the non-hemolytic HDP magainin could completely inactivate its antimicrobial activity. The loss of activity in this case was accompanied by the loss of the \(\alpha\)-helical structure. [PERSON] and [PERSON] [50] postulated that this finding may have occurred because magainin already had optimal combination of hydrophobicity and charge; thus, any alteration to the sequence only disrupted the delicate combination and adversely affected its activity. [PERSON] et al.