Proton Transport by Lugdunin Analogs

Proton transport induced by lugdunin and its analogs was analyzed using the pH-sensitive dye pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid). LUVs were filled with 100 mm KCl, 10 mm HEPES, and 0.5 mm pyranine (pH = 7.4), and extravesicular dye was removed after extrusion via size exclusion chromatography (Illustra NAP-25 G25, GE Healthcare, Chalfont St Giles, UK). The vesicles were diluted in the same buffer without pyranine and pH = 6.4 to a final lipid concentration of 50 µm. Pyranine fluorescence was monitored in a time-dependent manner with λ_ex = 458 nm, λ_em = 512 nm, and band widths of 3 nm using an FP 6500 spectrofluorometer (Jasco Germany, Groß-Umstadt, Germany; Spectra Manager V. 1.54.03) under constant stirring. After acquiring a baseline for 100 s, peptide stock solution in isopropanol was added to a nominal peptide-to-lipid ratio (n/n) of 1:250. Acidification of the lumen resulted in fluorescent quenching and was monitored over the course of 500 s. Afterward, the vesicles were lysed by the addition of N,N-dimethyl-n-dodecylamine N-oxide (LDAO) leading to a disruption of the pH gradient. All data points were normalized to the fluorescence intensity directly before the addition of the peptide and after vesicle lysis.