NBD and tryptophan fluorescence assay

NBD experiments were performed at 37°C in a 4 × 4 mm cuvette (volume 240 µl) in a FP 8300 fluorimeter (Jasco, Japan). Excitation was set at 505 nm (bandwidth 2.5 nm). Emission was recorded from 520 to 650 nm (bandwidth 5 nm). The cuvette initially contained liposomes of defined size and composition (0.15 mM lipids) in HKM buffer (Hepes 50 mM, pH 7.4, Kacetate 120 mM, MgCl₂ 1 mM, DTT 1 mM) and a blank spectrum was recorded. Thereafter, 0.125 µM NBD-labeled GMAP (1–375 fragment with the M1C mutation for NBD labeling [Pranke et al., 2011]) was added and another spectrum was recorded. The second spectrum was corrected for the blank.

For tryptophan fluorescence, excitation was set at 280 nm (bandwidth 5 nm) and emission was recorded from 300 to 450 nm (bandwidth 5 nm). The cuvette initially contained liposomes (DOPC/DOPE/Cholesterol 60/30/10 mol/mol; 0.5 mM lipids) of the indicated size in HKM buffer and a blank spectrum was recorded. Thereafter, 1 µM peptide (ALPS or condALPS) was added from 50X stock solutions in urea (4 M) and another spectrum was recorded and corrected for the blank.