2.2.3. Lipid Mixing

Lipid mixing of membrane fusion was measured by FRET using a Cary Eclipse (Varian) spectrofluorometer. For each lipid composition, unlabeled and labeled LUVs were prepared. To prepare the labeled LUVs, we included 1 mol % NBD-PE and N-Rh-PE in the lipid mixture before drying the lipids in the liposome preparation procedure. Unlabeled and labeled LUVs were mixed at a 9:1 ratio in pH 5.0 buffer. The total lipids concentration was 0.2 mM. After the equilibration of the vesicles, an appropriate amount of peptides from a stock solution was added to give final concentrations of 4 μM. Then, 10% Triton X-100 was added to achieve a final concentration of 1% after fusion had been completed. Fluorescence intensity of the acceptor (excitation with 463 nm and emission at 590 nm) before the addition of peptides and after the addition of Triton X-100 was defined as 0% and 100% fusion, respectively. Experiments were performed in triplicates and averaged signal is shown.

## 2.2.4. Tryptophan Fluorescence

Fluorescence measurements were made with a Carry Eclipse (Varian) spectrofluorometer with an excitation wavelength of 280 nm, excitation/emission (2.4/4 nm), and photomultiplier voltage of 800 V. Spectra were measured using a 4 × 10-mm cuvettes (Hellma) in of 295–500 nm emission region with an increment of 1 nm. Peptide solutions

were used in 2–10 μM concentrations in 1000 μL volume, titrated with increasing portions of SUV suspension up to 1 mM in 13–20 steps. Normally, for each lipid concentration, three spectra were averaged to achieve an adequate signal-to-noise ratio. The cuvette was in the contact with a thermostat, assuring constant temperature of 22.0 ± 0.5 ◦C. From each spectrum background was subtracted (by measuring blank titration). The titration curves were constructed as normalized intensity values for the wavelength for which the maximum spectral shift was observed between free and liposome-bound peptide (∼328 nm). Such procedure was shown to govern a linear response of the signal in respect to the titrated peptide [48]. The titration curves were further corrected for SUV scattering [48] by using the tryptophan (N-acetyl-L-tryptophanamide) fluorescence registered under the same conditions in the presence of SUV solution at concentration [L] according to the equation:

$$F\_{pept}^{corr}([L]) = F\_{pept}([L]) (F\_{Trp}^{buffer}) / (F\_{Trp}([L])) \tag{3}$$

To corrected data point, non-linear hyberbolic curve was fitted according to the equation:

$$F = 1 + (I - 1)(\mathcal{K}\_{\mathbf{x}}[L]) / ([\mathcal{W}] + \mathcal{K}\_{\mathbf{x}}[L])\tag{4}$$

where I denotes asymptotic intensity value, [W] is the molar water concentration (55.55 M), and *Kx* is the molar partition coefficient. Gibbs free energies were calculated as: Δ*G*◦*x* = −*RT* ln *Kx*.

## 2.2.5. Tryptophan Quenching

We measured the quenching of tryptophan residues inside lipid bilayers using lipids labeled with bromine at carbons 4–5, 6–7, 9–10 and 11–12 in the acyl chain (denoted as Br4,5, Br6,7, Br9,10, and Br11,12, respectively), with 20 mol% brominated PCs in place of POPC. The same spectral conditions and spectrofluorometer settings were used as in binding experiments. Aliquots of peptide solution were added to vesicle suspensions to achieve a final peptide/lipid ratio of 1:500 and incubated for 2 min before recording tryptophan fluorescence intensity. The signal from an identical sample without peptide was used to determine the quenching (*Fm*/*F*0).

## *2.3. Leakage Assay*

Leakage assay was performed by monitoring fluorescence increase of calceinrealeased from LUV interior. Calcein was encapsulated in LUVs by hydrating lipid film by dye solution (112 mM of calcein in 0.27 M NaOH). Free dye and liposomes were separated to fraction by size exclusion chromatography (PD-10 columns, GE Healthcare). The most concentrated fractions with liposomes were pooled. Final concentrations of lipids were 0.625 mg/mL. Fluorescence was recorded with Carry Eclips (Varian) using single excitation/emission wavelengths (495/518 nm). Fluorescence baseline of 3.65 μM liposomes was observed for 2 min (subsequently rescaled to 0% leakage). Then, the peptides were added to the solution at 1:50 peptide/lipid ratio and the leakage was measured over 4000 s. The remaining intact liposomes were disrupted by adding 10 μL of 10% Triton X-100 to the solution (100% leakage).
