## 4 Discussion
The difference in the importance of the net charge feature between the two datasets (peptides active against Gram-positive bacteria vs. peptides active against Gram-negative bacteria) may be due to the differences between the cell wall components of anionic characters. On the other hand, for the Gram-positive dataset, the isoelectric point (pI) was found to be the most important feature, while it was the second most important feature for the Gram-negative dataset. The pI is defined as the pH at which the net charge of a protein/peptide is equal to zero. In other words, a protein has zero net charge at its isoelectric point. As the pH of the environment becomes closer to the isoelectric point of the peptide, the net charge on the peptide surface gradually decreases and peptide-peptide interaction increases. Proteins have minimum solubility at or near their isoelectric point while protein solubility increases when pH moves away from pI. The pI is a feature that is closely related to the peptide charge and directly affects solubility. When the pH is equal to the pI of the peptide, the peptide loses its solubility and hereby its biological function [116]. Therefore, pI has an important role to exhibit the AMP's antimicrobial activity; pls of the AMPs are generally at alkaline pH, and hereby maintain their activity at physiological pH. Therefore, the isoelectric point is another important feature that administers the antibacterial activity of AMPs [117, 118, 119, 120]. [PERSON] et al. reported that rather than the net charge, pI was a better parameter for predicting the antibacterial activity [121]. Our results are in accordance with the previous literature, supporting the feature ranking analysis performed in this study. Along this line, the findings of this study support the idea that isoelectric point and the net charge are two main descriptors of antimicrobial peptides. In our experiments, the above-mentioned two features were followed by the disordered conformation propensity, normalized hydrophobicity, and normalized hydrophobic moment features, respectively, for both bacterial groups. The majority of LCAMPs are disordered structures in aqueous solution and acquire their biologically active conformation upon interaction with the membrane. The majority of linear AMPs adapt to the alpha-helical conformation in lipid membrane environment, and this regular structure is important for antimicrobial activity for this AMP class [122]. Hence, the identification of disordered conformation propensity feature as the third important feature in our analysis makes sense in terms of the underlying biology. Hydrophobicity and hydrophobic moment are two important physico-chemical features that affect the antimicrobial activity of AMPs. In this study, the effect of these determinants was found lower than expected. The hydrophobicity reflects the ratio of hydrophobic residues within a peptide sequence. In the first step of peptide-lipid interactions, AMPs attach to the cell surface by electrostatic interactions, and then the hydrophobic interactions become a primary driving force for their insertion and partitions into the lipid bilayer [123, 124]. In general, the increase of hydrophobicity promotes antimicrobial activity in peptides [125]. However, some studies demonstrated that an increase above a certain level in hydrophobicity leads to a decrease in antimicrobial activity [125]. The hydrophobic moment is defined as a quantitative measure of peptide amphipchiatry [126]. The amphipathic \(\alpha\)-helical AMPs have polar and hydrophobic residues that are arranged in opposite faces. This arrangement facilitates the interactions of AMPs to membranes. The increase of the hydrophobic moment results in a significant elevation in antimicrobial activity, however, it also leads to cytotoxicity [124]. In addition to the physico-chemical descriptors, we have comparatively evaluated the effect of structure-based and sequence-based features on the classification performance.