## 2 Results and Discussion
### Interactions of P13#1 with SDS, LPSs and Bacterial Cells
This emission, which is typical of the neutral form of luciferin (Figure S3 in Supplementary Materials), can only be observed in environments with very low water contents and no alternative proton acceptors, the sole conditions that can hinder the photoinduced deprotonation process [70], and confirms that the fluorophore is buried into the micelles. Furthermore, the blue shift of the phenolate emission (5-10 nm) confirms that the fluorophore is in a less polar environment. In the presence of the LPS micelles, the emission spectra show an even stronger blue shift of the phenolate emission (15-20 nm), but the blue emission is only visible in the case of Luc-P13#1, suggesting a deeper penetration into the micelles or a different orientation of the peptoid with respect to Luc-GKY20. In the presence of bacterial cells, we also found a large blue shift of the phenolate emission of 18-22 nm in the case of Luc-GKY20 and of about 15 nm in the case of Luc-P13#1. Once more, a small emission in the blue region was detected only in the case of Luc-P13#1, thus confirming the existence of small differences between the membrane-binding modes of Luc-GKY20 and Luc-P13#1. Very interestingly, the emission spectra were very similar in the presence of _E. coli, P. aeruginosa and S. aureus, with only small variations in the emission wavelength (less than 5 nm). In the case of aLuc-GKY20 and aLuc-P13#1 (Figure 7), slightly larger blue shifts were detected upon binding to SDS, the LPSs and the bacterial cells (shifts of 15-19 nm, 25-28 nm and 19-21 nm, respectively), as is expected for the more solvatochromic aminoluciferin. Figure 6: Emission spectra after excitation at 330 nm of Luc-GKY20 and Luc-P13#1 in the presence of SDS, LPSs or bacterial cells. (**A**,**B**) Emission spectra of Luc-GKY20 and Luc-P13#1, respectively, in NaP (blue line), SDS (red line) and LPSs (green line). (**C**,**D**) Emission spectra of Luc-GKY20 and Luc-P13#1, respectively, in NaP (blue line) or in the presence of _E. coli_ (red line) _P. aeruginosa_ (green line) and _S. aureus_ (yellow line) cells. In each panel, the emissions in the presence of SDS, LPSs and bacterial cells were normalized to the emission in NaP. The binding of the P13#1 variants to SDS, the LPSs and the bacterial cells was also accompanied by strong positive or negative variations in the emission intensity. Usually, solvatochromic fluorophores show higher emission intensities in apolar environments, but luciferase are also sensitive to quenching, e.g., intramolecular, conformation-dependent quenching from tryptophan and tyrosine [70]. Therefore, the observed variations in the fluorescence emission are a net result deriving from these two opposite contributions. The reduction in the intensity of the emission was particularly evident in the presence of bacterial cells, likely because bacterial membranes contain proteins and other non-lipidic compounds (e.g., pigments) that might contribute to quenching (intermolecular quenching). We also studied the variations in the emission spectra of aLuc-GKY20 and aLuc-P13#1 in the presence of bacterial cells as functions of time (Figures S4 and S5 in Supplementary Materials). In all the cases, we found that the binding process was complete within 5 min. Therefore, the interactions of labeled GKY20 and P13#1 with bacterial cells are similar also from a kinetic point of view. As LPSs are very strong proinflammatory molecules, CAMPs with high levels of affinity for these endotoxins show anti-inflammatory activities, acting as LPS scavengers. Therefore, we used Luc-P13#1 to determine the K\({}_{\mathrm{D}}\) values for submicellar (5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) and micellar (40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) LPSs from _E. coli_ O111:B4 or _P. aeruginosa_ 10. Fluorescent assays were carried out via incubating increasing concentrations of the labeled P13#1 and GKY20 with a constant concentration of LPSs.