# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 4 Mechanism of Membrane Destabilization  
A number of dedicated studies on the mechanism of membrane destabilization made use of model membranes for determining whether diastereomeric HDPs differ from their all-l enantiomeric counterparts or not. The vast majority of mechanism studies performed so far have been conducted using linear peptides with helical propensity against bacterial targets. This section summarizes and outlines the mechanisms by generalizing the available information from the literature. At the conceptual level, d-AA-containing HDPs recognize their targets in a manner identical to that of all-l enantiomeric HDPs. The electrostatic attraction of the positively-charged peptide with negatively-charged moieties on the target membrane forms the first step by which the d-AA-containing peptides recognizes and binds the membrane [49; 50; 51]. Particularly, diastereomers and all-d enantiomers have been tested in this first step against all cellular pathogens, namely bacteria [1], cancer [13], fungi [3] and parasites [4]. For viruses, there is little information on how HDPs recognize and bind to the target. There is an indication that electrostatic binding does have a role in viral recognition. The negatively-charged sialic acids found on a number of viruses were shown to attract HDPs and are possibly responsible for the HDP recognition and binding, which was demonstrated with a scorpion venom peptide recognizing a herpes simplex virus [5]. No information could be found on whether there is a difference in the way diastereomer and all-l HDPs recognize viruses. Although electrostatic attraction is the overall governing feature in HDP target recognition, there are important subtle differences in how diastereomers bind negatively-charged membranes. [PERSON] and [PERSON] [49] performed a dedicated study with the aim of elucidating the mechanism behind the often profound difference in the selective toxicity of diastereomers compared to that of all-l enantiomer AMPs. The study was performed using model membrane consisting of phosphatidylethanolamine and phosphatidylglycerol (PE/PG) that mimicked a negatively-charged bacterial membrane while the neutral mammalian membrane was represented by phosphatidylcholine and cholesterol (PC/Cho). A schematic representation of the membrane interaction mechanism described in their study is summarized in Figure 2.  
Binding analysis measured by surface plasmon resonance on a lipid heteromonolayer and bilayer revealed that L-AA peptides binds similarly to both PE/PG and PC/Cho membranes. However, their corresponding diastereomers binds 50- to 100-folds better to negatively-charged PE/PG membranes than to neutral PC/Cho membranes. Further analysis showed that the all-L peptides binds 10- to 25-folds stronger to zwitterionic PC/Cho bilayers than to monolayers, whereas the diastereomers binds similarly to bilayer and monolayer membranes, indicating that the inner layer of zwitterionic membranes contributes significantly to the binding of the all-L peptides but not of their diastereomers. Figure 2: Membrane interaction schematic of diastereomer and all-L peptides with zwitterionic and negatively-charged membranes. Step I: both types of HDP bind to membranes via the electrostatic force, and in the case of all-L peptides, a hydrophobic interaction is also present. Step II: increasing the peptide concentration will result in different outcomes depending on the peptide and membrane type. (**A**) Diastereomer interaction with a zwitterionic phosphatidylcholine and cholesterol (PC/Cho) membrane. The peptide remains at the surface and does not disrupt the membrane structure significantly; (**B**) The carpet mechanism, the primary diastereomer interaction pathway with the negative phosphatidylcholine and phosphatidylcholine/polycerol (PE/PG) membrane. A large number of the peptides engulf the membrane like a carpet, and their amphipability induces micellization.