Supplementary Figure 1: Sample characterization.

1. FRET assay indicates no aggregation of KvAP-VSD in both PC:PG and DOTAP liposomes. (a) Cartoon representation of FRET assay to evaluate the aggregation behavior. KvAP-VSD mutant A111C (b) was individually labeled with fluorescence donor and acceptor, then mixed at 1:1 molar ratio and reconstituted into liposomes. FRET signal in the range of 560-580 nm indicates of closeness of fluorephores in liposome, thus the degree of protein aggregation (see Methods for details). (c) Amplitude normalized FRET spectra of KvAP-VSD in liposomes with incremental DOTAP contents. The intensity within region 560-580 nm is essentially the same among all tested conditions, which suggest no severe aggregation of KvAP-VSD in both POPC:POPG and DOTAP liposomes. (d) Conformation dependent polarity change upon increment of DOTAP content shown by the wavelength shift of fluorecin emission (grey region, (c)). The sigmoidal fit agrees very well with the titration results monitored by spin labeling methods (Fig. 2). 2. DOTAP liposome is physically equivalent to POPC:POPG liposome regarding to oxygen and NiEdda accessibilities. (e) EPR spectra of nitroxide radicals at various depths in both PC:PG and DOTAP liposomes, probed by radical containing phosphocholine lipids (see Methods for details). (f) No NiEdda accessibility (ΠNi) was observed at all studied positions in both liposomes. (g) Oxygen accessibility (ΠO₂) was centralized in the middle of lipid bilayer and decreased toward the surface of liposomes. Both liposomes have the same pattern with slightly different amplitudes.