Figure 1
Ligand-induced changes in A2AR conformations measured by smFRET. (a) A double-cysteine mutant of A2AR (T119C Q226C) labelled with AF488 (donor) and AF647 (acceptor) was alternately excited with blue (473 nm) and red (635 nm) lasers; bursts of fluorescence of the donor (green) and the acceptor (red) were recorded from single receptors diffusing through the confocal detection volume. The FRET efficiency E for each burst was calculated using Eq. 1a and the values obtained from all bursts for each ligand condition were used to build smFRET histograms (c–f). (b) Width (ΔEFWHM) vs. mean (< E >) for the FRET efficiency distributions shown in (c–f); the error bars were estimated by statistical bootstrapping. Shown as reference are the regions of the shot-noise limit (blue) and of the quasi-static limit (dsDNA, pink) (see SI Sect. 1.2 for details). (c–f) smFRET distributions of A2AR in the (c) basal state (apo), and in the presence of (d) 100 μM ZM241385 (antagonist), (e) 100 μM LUF5834 (partial agonist), and (f) 100 μM NECA (full agonist), fitted to a sum of Gaussians (black line). Solid blue, green and red lines represent individual Gaussians and corresponding dashed vertical lines represent their peak center. The results of the fitting are shown in Table 1.
