NBD fluorescence assay

We conjugated the environmentally sensitive dye NBD to the C-terminal cysteine in pHLIP using n, n′-dimethyl-n-iodoacetyl-n′-7-nitrobenz-2-oxa-1,3-diazol-4-yl ethylenediamine (IANBD; Thermo Fisher Scientific, Waltham, MA). For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6-n-7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA). To this end, a pHLIP variant lacking a cysteine residue (N_t-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-C_t) was employed. After purification by size exclusion chromatography using a PD-10 column (GE Healthcare Bio-Sciences, Marlborough, MA) in 1 mM NaP_i buffer, pH 7.5, the conjugation was verified by MALDI-TOF and high-performance liquid chromatography, and samples were aliquoted and lyophilized. For the experiment, samples were rehydrated in 10 mM NaP_i pH 8.0 at a final concentration of 0.8 μM (the NBD extinction coefficient was 18,482 M⁻¹ cm⁻¹) and incubated with POPC vesicles (prepared as described earlier) for 1 h. All experimental conditions, including lipid/peptide and final salt concentration (19.3 mM), were identical to W fluorescence and CD experiments. The pH of each row of a 96-well plate was decreased using a series of 100 mM buffers (sodium acetate and NaP_i (14.3 μL)). Final sample volume was 100 μL. Fluorescence spectra were recorded at 25°C with excitation at 470 nm and an emission range of 520–600 nm using a Cytation 5 imaging plate reader (Biotek Instruments, Winooski, VT). The final pH of each well was measured. Fluorescence emission intensity pH-titrations at 540 nm were then fitted to determine the pK_Ct-NBD and pK_Nt-NBD using Eq. 2.