# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities
### Antimicrobial Activity
[51] provided insights into why diastereomers are able to increase the perforation of negatively-charged membranes despite their low tendencies for hydrophobic interactions due to unstable secondary structures (i.e., despite their low tendencies for hydrophobic interactions due to unstable secondary structures) by inserting into the hydrophobic core [49]. Combined Nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, fluorescence quenching and lipid/polylaacetylene colorimetric analyses revealed the following: (i) although the tested diastereomers were nearly devoid of helical structures however diastereomer peptides were able to segregate polar and hydrophobic faces from one another and (ii) helical structures are essential for l-AA peptides to be able to orient their hydrophobic and polar amino acids into separate faces thus allowing hydrophobic interactions with membranes. This separation allowed the diastereomers to insert into the hydrophobic membrane core. The diastereomers in this case were unstructured in solution but were able to adopt a helical structure upon membrane binding. Moreover, their results also indicated that the interaction between the positively-charged diastereomer and the negatively-charged bacterial membranes allowed the peptides to overcome the energy barrier and adopt a stable secondary structure. This finding provides an explanation for the unanswered question of why diastereomers are capable of hydrophobic interaction with bacterial membranes but not mammalian ones [49]. Another effect of the reduced hydrophobicity and amphipabticity resulting from the disruption of secondary structure by d-AAs is the reduction of the peptide aggregation potential in solution [18; 63]. Reverse-phase HPLC temperature profiling revealed that high peptide hydrophobicity and amphipabticity is associated with greater peptide self-association in solution, which also correlates with toxicity against mammalian cells [18]. Furthermore, the authors of this study argued that the capacity to decrease peptide self-association in aqueous media also decreases the toxicity against mammalian cells because it allows the peptides to dissociate more easily from each other and associate with the membrane interface, thus preventing further penetration into the hydrophobic core. In contrast, for a negatively-charged bacterial membrane, the interaction of monomeric peptides with the phospholipid head group at the membrane interface was solely sufficient for destabilization. Additionally, no extensive insertion into the membrane core was required such as in the case of the barrel-stave mechanism. In addition to solution aggregation, [PERSON] and [PERSON] [63] noted that the capacity to form aggregates inside the membrane was positively correlated with the hemolytic strength of the diastereomers, thereby underscoring the detrimental effect of peptide aggregation on the selective toxicity of the peptide. Furthermore, the results of [PERSON] et al. [18] showed that amongst the different factors (e.g., hydrophobicity and amphipabticity) governing the selectivity of antimicrobial HDPs, helicity had the highest influence. It was found that high helicity was correlated with strong hemolytic activity whereas a reduction in helicity, for instance by the introduction of d-AAs, would correspondingly reduce hemolysis while generally maintaining the antimicrobial activity. It should also be noted that a stable helical structure was not necessary for high selectivity index [57; 61; 63; 64]. The lack of a stable secondary structure and the low amphipabticity of diastereomers suggests that the carpet mechanism and not the barrel-stave mechanism is a requirement for strong and selective antimicrobial activity [18; 65].