Peptide synthesis

Peptides were synthesized by a solid-phase method using the Fmoc methodology on an automated microwave peptide synthesizer (Liberty-1, CEM, Orsay, France) as previously described^29,45. H-Rink amide ChemMatrix resin (100 micromoles, substitution 0.37 mmol.g⁻¹, Longjumeau, France) was used to generate an amide function at the C-terminal end of all peptides. The MgtR peptide (MNRSPDKIIALIFLLISLLVLCLALWQIVF), MgtR-S17I single variant, MgtR-S17I-C22S double variant and MgtR-S17I lacking WQIVF at its C-terminal end (MgtR-S17I short) were synthesized following a double-coupling step for each amino acid (400 micromoles) activated with TBTU (500 micromoles). Additionally an acetylation step was applied at the end of each amino-acid incorporation to prevent the formation of incomplete peptides. After clivage from the resine and precipitation⁴⁵, the final product was resuspended in an isopropanol/water (50%/50%: vol/vol) without any purification and analysed by MALDI-TOF mass spectrometry (Fig. ^S4). MgtR and MgtR-S17I peptides were synthesized two times independently and similar results were obtained from both synthesis. For experimentations, peptides were resuspended at a concentration of 3.2 mM in DMSO/water (50%/50%: vol/vol) as reported⁴⁵. Using the same procedure, we also synthesized a scrambled peptide based on the amino-acid sequence of MgtR (permutation of the original peptide, ILFVADSLQMIPLCLRIWVALKINILFSVL)⁴⁵.

For N-terminal labelling of the peptide with fluorescein, a spacer made with an aminohexanoic acid was first coupled to the N-terminal amino group as recommended⁴⁸ to prevent the subsequent cyclization of the fluorescein with removal of the first amino acid. After coupling of the fluorescein, the peptide was processed as described above for the unlabeled peptide.

For C-terminal labelling of the peptide with fluorescein, a Fmoc-Lys(Aloc)-OH amino acid was first coupled on the resin as the C-terminal amino acid and the peptide was then synthesized as previously described. The Fmoc group was kept at the N-terminal end of the peptidyl resin until the labelling of the C-terminal end with fluorescein. At the end of the synthesis, the Aloc group was thus orthogonally removed by treating the resin following the tetrakis/phenylsilane procedure as previously described⁴⁹. Once deprotected, the labelling of the epsilon-amine group of the C-terminal lysine was performed with a 10 fold excess of FITC and the resin was allowed for labelling overnight. Fmoc group was then removed and the peptide was dried prior final deprotection and cleavage step using standard conditions as described above.