Sly1 helix α21 binds membranes, with a preference for higher curvature. TMR-α21 and TMR-pα21 peptides were added to liposomes of nominal diameter 30 and 200 nm, which contained 1% TRPE as a fluorescence acceptor. (A) Emission spectra of peptides or liposomes (30 nm diameter, 6.7% ergosterol) measured separately, and the sums of the peptide and liposome spectra. The sums represent the no-FRET condition. Both the TMR-α21 and TMR-pα21 spectra are plotted; they overlap almost exactly. Vertical dashed lines at 585 and 610 nm indicate emission peaks for labeled peptides and liposomes, respectively. (B) Example of FRET data. Spectra from binding reactions containing liposomes (30 nm diameter, 6.7% ergosterol, 500 µM total lipid) and 25 µM TMR-α21 or TMR-pα21 are shown. The no-FRET condition is shown for reference. (C) Normalized FRET ratios for binding reactions containing the indicated combinations of liposomes and peptides, as in panel B. Traces and bars in A–C show means and ±95% confidence bands from four independent experiments.