*3.2. Conformational Analysis of Lacticaseicin 30*

The secondary and three-dimensional (3D) structures of lacticaseicin 30 were predicted using AlphaFold2 [36], a machine learning approach that was proven to deliver highly accurate and reliable protein structure prediction [46]. As seen on Figure 2, the predicted secondary structure of lacticaseicin 30 is characterized by the presence of five helices (73 amino acids out of 111, i.e., 66%), several connecting coiled regions (38 amino acids out of 111, i.e., 34%) and five potential H-bonds indicating interactions between these distinct helical segments. The predicted largely helical structure was further confirmed by circular dichroism (CD). CD spectra were obtained at pH 7 and 5, in the presence or absence of sodium dodecyl sulfate (SDS) micelles used as a model of the anionic membrane bilayer [47]. The CD spectra showed that lacticaseicin 30 adopts a helical structure at both pH 5 and 7, and in the absence or presence of SDS micelles (Figure 3A). The content of the different secondary structures, in terms of helix, antiparallel and parallel β-sheets and turns, was evaluated using the BestSel software (Figure 3B). The percentage of α-helix in the presence of SDS micelles was 29% and 63% at pH 7 and 5, respectively, while it was estimated at 41% and 54%, respectively, in the absence of SDS micelles. These results show that interaction with a membrane-like environment is not required to trigger the organization of lacticaseicin 30 in a helical structure, which is intrinsically acquired even in buffer. Moreover, they show that an acidic pH environment favors a higher proportion of helix, suggesting it helps in folding the active structure of lacticaseicin 30 (Figure 3B).

**Figure 1.** Mass spectrometry analyses of 6his-tag-depleted Ser-lacticaseicin 30 (average molecular mass 12,339.2 Da). (**A**) Linear positive MALDI-TOF mass spectrum illustrating the [M + H]+ and [M + 2H]2+ mass signals obtained. The measured MALDI-TOF-MS molecular mass was evaluated as 12,349.2 Da. (**B**) ESI mass spectrum and deconvoluted molecular mass (UniDec DScore: 54.16) revealing a molecular mass of 12,340 Da. (**C**) Peptide mapping of the 6his-tag-depleted Ser-lacticaseicin 30 after tryptic hydrolysis. Blue lines correspond to identified peptides along the lacticaseicin 30 sequence following the bioinformatic retreatment of RP-HPLC-MS/MS data using PEAKS Studio Xpro and the UniProtKB/Swiss-Prot-TrEMBL protein databases restricted to *Lactobacillus* (https://www.uniprot.org/, accessed on April 2022).
