Fluorescence extinction of LacY in the presence of brominated phospholipids

The fluorescence measurements were conducted using a QuantaMaster model QM3-SS (Photon Technology International), a cuvette-based fluorescence spectrometer. Data were collected and analyzed using Felix 32 software. All measurements were conducted in degassed 50 mM Tris-HCl (pH 7.5)/100 mM NaCl/0.05% DDM. Using a Peltier TE temperature controller, the sample was held at a constant 20 °C. The excitation wavelength was fixed at 295 nm, and emission spectra were recorded from 310 to 370 nm, with a bandwidth of 1 nm used for both excitation and emission beams. In the fluorometer stirred cuvette, DDM-solubilized LacY derivatives were suspended at 50 μM and equilibrated at 20 °C. Small volumes of detergent or lipid together with detergent were then added from various stock solutions of detergent micelles or mixed micelles. DDM micelles (50 mg/mL) containing either no lipid (DDM only), DOPC at 10 mg/mL or (9–10Br)-PC at 10 mg/mL or 20 mg/mL were incubated 2 h in the presence of 50 μM protein. Incorporation of phospholipid into the LacY-containing DDM micelles was measured by detecting the quenching of LacY intrinsic tryptophan fluorescence at 330 nm. The fluorescence extinction of Trp was evaluated using the ratio F/F₀, where F and F₀ are the fluorescence at 340 nm in the presence of mixed micelles containing or not containing brominated lipids, respectively. Results represent the average of five independent measurements obtained from emission spectra that were corrected for measurements with DDM only.