## 2 Results and Discussion  
### The Design of Peptide P13#1  
Peptoid P13#1 was designed taking into consideration our previous work on the identification of cryptic CAMP-like sequences [39]. This method has allowed for the identification of many human peptides [40; 41; 42; 43] and, more recently, the identification of dozens of new CAMPs via screening the whole human proteome [44]. We showed that a correlation does exist between the antimicrobial potency (defined as log(1000/MIC)) of a peptide and a score dependent on the net charge and hydrophobicity of the peptide and two strain-dependent constants. The relationship is linear for scores between about 6 and 10-11, whereas for scores higher than 11, an increase in the score does not necessarily increase the potency of the peptide. Even if the \"ideal\" composition of a CAMP depends on a specific bacterial strain, our analysis suggested that on the average, a high-potency CAMP should contain about 40% basic residues (Lys and Arg) and 60% of the most hydrophobic residues (Trp, Phe and Leu). In particular, a peptide with six Lys/Arg residues and seven Trp/Phe/Leu residues would have a score between 11 and 13, depending on the specific set of parameters used for the calculation (the hydrophobicity score list and strain-dependent constants), thus being the shortest peptide with score higher than the linearity window. Therefore, to verify whether data acquired from the analysis of CAMPs can be translated to peptoids as well, we decided to synthesize a peptoid with six basic and seven aromatic residues. As a basic residue we preferred NLys, the peptoid analogue of lysine, over \(N\)Arg because the use of an \(N\)Lys residue facilitates the synthetic process; moreover, according to our analysis, in high-scoring peptides, Lys and Arg are equivalent. As an aromatic residue, we selected \(N\)-(2-phenylethyl)-glycine (\(N\)pet), the peptoid analogue of homophenylalanine, as this residue is slightly more hydrophobic than \(N\)Phe (the peptoid analogue of Phe) and is more stable and easier to handle with respect to the peptoid analogues of Trp [45]. Finally, we selected a specific sequence of six \(N\)Lys residues and seven \(N\)pet residues (Figure 1A), providing an amphipathic structure both when folded as (all-_cis_) polyproline-I-like and (all-_trans_) polyproline-II-like helices, as discussed below. One of the \(N\)pet residues was changed to \(N\)-[2-(4-methoxyphenyl)ethyl]-glycine (\(N\)mpe) in order to obtain a peptoid with a good extinction coefficient at 280 nm, thus simplifying the purification and quantification steps. It is worth noting that the sequence of P13#1 shows some similarities with the sequences of bovine indolicidin and porcine tritrpticin, the two shortest and yet very active mammalian cathelicidins [33], even if P13#1 was not specifically designed to mimic these two CAMPs (Figure 1B). Monte Carlo molecular modeling was used to perform an in silico conformational analysis. Two implicit solvation functions were used to predict the conformation of the peptoid in water and in a membrane-mimicking environment (octanol), respectively. Using octanol solvation, the more extended polyproline-helix-II-like conformation (Figure 2A) was found to be about 2.9 kcal/mole more stable than the polyproline-helix-I-like conformation (Figure 2B). Even more interestingly, long minimizations did not change the regular helical conformations, thus indicating that in a membrane-mimicking environment, the two conformations represent very distinct local minima. This is because the effective solvation of the hydrophobic side chains by octanol prevents the formation of intramolecular contacts among them which, in turn, could stabilize the more compact conformations. We want to underline that as P13#1 has no stereogenic centers, each helical conformation can exist both as isoenergetic, enantiormophous left- and right-handed helices (Figure 2). This implies that in solvents (or any achiral environment) favoring helical conformations, left- and right-handed helices will coexist.