Fig. 3: Time-resolved anisotropy show fast rotational correlation dependent on fluorophore position.

a Photon arrival time histograms of vertically (green) and horizontally (light green) polarized photons arising from a CHO-K1 cell transiently transfected with N-terminal-SNAP-β₂-AR conjugated to Alexa Fluor 488 dye. The instrument response functions (IRF) for vertical and horizontal polarization are shown in gray and dark gray, respectively. The histograms were fit globally with Eqs. (7) and (8) to obtain a slow rotational correlation time (τ_rot) of 39 ns. The fits are shown in black. The bottom panel gives the reconstructed anisotropy decay and the corresponding fit. Weighted residuals corresponding to the global fits are shown on top. b Rotational correlation times of all β₂-AR constructs, naming and color scheme as in Fig. 2. NT shows 113.7 ± 70.5 ns; IL3 shows 165.1 ± 14.6 ns; S shows 40.0 ± 27.5 ns and TAG shows 118.3 ± 51.8 ns. Please note that from biexponential rotational correlation time fitting, the slower τ_rot was used. Data are mean ± s.d. c Steady-state anisotropy (r_ss) is largely independent from receptor density within the measured concentration range of all β₂-AR constructs. Squares represent NT, circles represent IL3, triangles represent S and diamonds represent TAG.