Indicated concentrations of His10 proteins were premixed and then incubated with DGS-NTA-Ni containing liposomes for 40-60 min to allow for membrane attachment. Following 40 min incubation, His10 proteins were undetectable in the supernatant after liposome sedimentation, suggesting complete membrane attachment.
FRET assays were carried out at RT in solid-white 96-well polystyrene plates (Corning) in a total volume of 100 μl per reaction. All protein components (including 0.3 mg/ml BSA in order to prevent non-specific binding) were mixed, followed by addition of liposomes, and incubated for 40-60 min, during which the SNAP-cell-505 fluorescence was monitored at the minimal intervals (6 s to 30 s, depending on the number of wells monitored) with 504 nm excitation and 540 nm emission. During this period, His10 proteins bind to the liposomes and the temperature equilibrates, establishing a stable baseline. ATP was subsequently injected followed by 5 s of automatic shaking of the plate, and the fluorescence was further monitored for at least 1 h. Data were normalized by setting the average fluorescence value of the last 10 data points before ATP addition as 100%, and background fluorescence as 0%.
FRET assays were carried out at RT in solid-white 96-well polystyrene plates (Corning) in a total volume of 100 μl per reaction. All protein components (including 0.3 mg/ml BSA in order to prevent non-specific binding) were mixed, followed by addition of liposomes, and incubated for 40-60 min, during which the SNAP-cell-505 fluorescence was monitored at the minimal intervals (6 s to 30 s, depending on the number of wells monitored) with 504 nm excitation and 540 nm emission. During this period, His10 proteins bind to the liposomes and the temperature equilibrates, establishing a stable baseline. ATP was subsequently injected followed by 5 s of automatic shaking of the plate, and the fluorescence was further monitored for at least 1 h. Data were normalized by setting the average fluorescence value of the last 10 data points before ATP addition as 100%, and background fluorescence as 0%.