Appendix 1—figure 4. Fluorescence decays of the purified sensor measured by FLIM.
Representative fluorescence decays of NADP-Snifit in buffer (50 mM HEPES, 150 mM NaCl, pH 7.5, 0.5 mg/mL BSA at 37 °C) measured by TCSPC-FLIM. The donor only sample corresponds to purified sensor labeled only with CP-TMR-SMX (red line). The fluorescence decay is fitted with a biexponential model (χ2red = 1.11), yielding an amplitude-weighted average lifetime <τ> of 2.84 ns. The FRET samples are prepared with the addition of 1 mM NADP+ (green line) or 2 mM sulfapyridine (blue line) in the aforementioned buffer to obtain, respectively the closed sensor with highest FRET efficiency (Emax) and the closed sensor with the lowest FRET efficiency (Emin). The fluorescence decays of FRET samples are best fitted with a 3rd-order exponential model. The closed and open sensor conformation yield <τ> of 1.03 (χ2red = 1.15) and 2.4 ns (χ2red = 1.07), respectively. Emax and Emin correspond to 64% and 15% according to the following equation: ; where τDA is the lifetime of the FRET sample (donor + acceptor) and τD is the lifetime of the donor-only sample.