N 7 nitrobenz 2 oxa 1 3 diazol 4 yl 1 2 dihexadecanoyl sn glycero 3 phosphoethanolamine nbd pe

The peptide sequences derived from the gp41 MPER-TMD region displayed in Figure 1b were produced as C-terminal carboxamides by solid-phase synthesis using Fmoc chemistry and purified by HPLC. PC-based synthetic phospholipids and dodecylphosphocholine (DPC) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) was from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Abberior STAR RED (KK114) was obtained from Abberior (Göttingen, Germany). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

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).