Fig. 5. Subtle conformational changes captured using the SeF-Bodipy593/FlAsH FRET pair. (A) Accessible volumes (AVs) of various dyes (Cy3, Cy5, SeF-Bodipy593, and FlAsH)⁵⁶ generated using software for FRET-restrained positioning and screening (FPS). (B) Conformation distributions of βarr1-T6C/L191C-(Cy3/Cy5), dashed lines r1 (black) indicate the mean FRET values. (C) βarr1-T6SeF-Bodpy593/L191-FlAsH before (top panel) and after (bottom panel) pp-β₂V2R addition, measured by smFRET. Dashed lines highlight the mean FRET values of βarr1-T6SeF-Bodpy593/L191-FlAsH before (R1, green) and after (R2, blue) pp-β₂V2R activation. R₀ of Cy3/Cy5 is 60 Å, and R₀ of SeF-Bodipy593/FlAsH is 60 Å. Data are shown as mean ± s.e.m. (B) and (C) show the representative data of three independent experiments. The FRET trace numbers for smFRET efficiency histograms: B (top): 1487, B (bottom): 1187, C (top): 3018, C (bottom): 2920.