Fluofit

VIPP1 (Preps #4, #5, and #6) and liposomes were mixed at 4 °C with protein and lipid concentrations of 0.4 mg/mL and 0.15 mg/mL, respectively, and incubated for 20 min at 22 °C before being transferred to a 3 mm × 3 mm quartz glass cuvette (Hellma Analytics) for measurement. TRF anisotropy measurements were recorded at 22 °C on a FluoFit 300 fluorescence lifetime spectrometer (PicoQuant) using time-correlated single photon counting. The excitation source was a pulsed LED emitting at λ_exc = 281 nm ± 5.5 nm. Emission was detected at λ_em = 355 nm ± 5 nm. Fluorescence decays were recorded with horizontal and perpendicular orientations of polarizers in the excitation and emission paths, respectively, each for 5 min. Photons were counted for 100 ns after each pulse in 4000 channels of 25 ps. The measurements were repeated after the addition of Mg²⁺ to a final concentration of 10 mM and an incubation at 22 °C for 20 min. Fluorescence anisotropy decays were fitted by a bi-exponential decay curve with FluoFit (PicoQuant) using data from 3–40 ns after excitation. Two rotational correlation times were obtained, where the fast one, corresponding to segmental tryptophan motions, was essentially constant at values < 0.5 ns, while the slow one, reflecting larger-scale protein motions, was exploited to probe VIPP1 binding to liposomes.