Lissamine rhodamine b 1 2 dihexadecanoyl sn glycero 3 phosphoethanolamine n rh pe

The peptides were custom ordered with purity >95% (Genemed Synthesis, Inc., San Antonio, TX, USA and Lipopharm, Gdańsk, Poland). Sequences were as following: HAfp1-23N: GLFGAIAGFIEGGWQGMVDGWYG-amide, HAfp1-20N: GLFGAIAGFIEGGWQGMVDG-amide. Acetylated peptide (Ac) had the N-terminal amine substituted by amide group. Stocks were prepared from weighted amounts dissolved in DMSO as 300–500 µM solutions. Concentrations were checked by UV spectroscopy using the extinction coefficient at 280 nm of 12.490 M⁻¹ cm⁻¹ for all peptides. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and C6-NBD-PC (1-palmitoyl-2-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)hexanoyl)-sn-glycero-3-phosphocholine)were purchased from Avanti Polar Lipids Ltd. (Alabaster, AL, USA) and used with no further purifications. (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-C6-PE) and Lissamine™ Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (N-Rh-PE) used in fusion assays were from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were from Sigma Aldrich (Saint Louis, MO, USA). All experiments were performed in buffer pH 5.0 (10 mM citric acid/NaOH, 150 mM NaCl), and additional binding experiments at pH 7.4 in 10 mM Hepes/NaOH, 150 mM NaCl.

Materials were custom ordered with purity $>95\%$ (Lipopharm, Gdańsk, Poland). Sequences were as follows: wt GLFGAIAGFIEGGWQGMVDGWYGSGKKKKD and its E11A and W14A mutants. In all cases, the N-terminus was unmodified and the C-terminus was an amide. The -SGKKKD sequence was introduced to increase peptide solubility. Stocks were prepared from weighted amounts dissolved in water as 300–500 M solutions. Concentrations were checked by UV spectroscopy using the extinction coefficient at 280 nm of 12490 M ${}^{-1}$ cm ${}^{-1}$ for wt and E11A peptides and 6990 M ${}^{-1}$ cm ${}^{-1}$ for W14A. N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) and Lissamine™ Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (N-Rh-PE) used in fusion assays were from ThermoFisher Scientific. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), Br ${}_{4,5}$ (1-palmitoyl-2-stearoyl-(4,5)dibromo-sn-glycero-3-phosphocholine), Br ${}_{6,7}$ (1-palmitoyl-2-(6,7-di-bromo)stearoyl-sn-glycero-3-phospho- choline), Br ${}_{9,10}$ (1-palmitoyl-2-(9,10-dibro-mo)stearoyl-sn-glycero-3-phosphocholine), Br ${}_{11,12}$ (1-palmitoyl-2-(11-,12-di-bromo)ste-aroyl-sn-glycero-3-phos-phocholine), and all other chemicals were from Sigma Aldrich (Merck, St. Louis, MO, USA). All experiments were performed in buffer pH 5.0 (10 mM citric acid/NaOH, 150 mM NaCl).