Circular dichroism

Measurements were performed on a Jasco (Easton, MD) J-815 spectropolarimeter at 25°C. pHLIP was incubated with POPC vesicles (prepared as described earlier) in 10 mM NaP_i pH 8.0 buffer for 1 h. Afterwards, the pH was adjusted with 100 mM sodium acetate, 2-N-morpholino ethanesulfonic acid, or NaP_i (62.5 μL) to a range of desired final pH values. Final sample volume was 250 μL. The lipid/peptide was 200:1 with a final peptide concentration of 7 μM. Changes in helical content were reported by following the ellipticity at 222 nm. Spectra were collected from 222 to 262 nm in 10-nm steps, and values at 222 nm were subtracted from values at 262 nm to correct for any changes in the baseline of each individual spectrum. By limiting the collected spectral range, the complete pH titration could be conducted in 5 h while acquiring data with high accuracy (each sample was averaged 20 times using a scan rate of 100 nm/min). Calculated molar ellipticity ([θ] = θ/(10 lcN) at 222 nm was plotted against measured pH, and the resulting sigmoidal transition was fitted using Eq. 2 to obtain the pK_CD.