Fig. 1: smFRET dynamics of individual A2AARs in the ABEL trap.
a A2AARs were labeled with donor (Alexa488, blue circle) and acceptor (Atto643, red circle) fluorescent dyes at the intracellular tip of the transmembrane helix TM6 and the amphiphilic C-terminal helix H8 and were reconstituted in lipid nanodiscs (gray belts). The FRET efficiency in labeled A2AAR increases upon receptor activation. b Sketch of the ABEL trap setup with four electrodes in the PDMS-based microfluidic chamber. The FRET donor fluorophore is excited by a fast-switching laser beam pattern, and the fluorescence of both donor and acceptor on individual receptors is collected through the objective of a custom-built confocal microscope. c The timescales of the structural dynamics observed using smFRET in the ABEL trap and the most common smFRET experiment modalities, i.e., confocal microscopy with freely diffusing molecules and TIRF camera-based microscopy with immobilized molecules. d Fluorescence traces in the donor (blue) and acceptor (red) channels were recorded for individual A2AARs held in solution by the ABEL trap. Background-corrected fluorescence traces were binned in 1-ms time-bins (thin semitransparent lines) and then smoothed using Chung-Kennedy filtering (thick lines). Fluorescence bursts with both donor and acceptor fluorescence are depicted with asterisks. e The histogram of the durations of the individual fluorescence bursts recorded for the apo A2AARs. The photon bursts shorter than 100 ms were excluded from the analysis (gray area). Out of the total 4080 bursts for apo A2AARs, 1948 bursts longer than 100 ms were selected and further filtered based on fluorescence intensity, yielding 1815 bursts for further analyses (see Supplementary Fig. 3, Supplementary Table 1). Exemplary fluorescence traces for the individual apo A2AARs (f–h) and A2AARs with agonist NECA (i–k). Donor (blue) and acceptor (red) fluorescence signals were plotted after binning with a 1-ms time-window and background subtraction (thin semitransparent blue and red lines). The areas outside of the selected fluorescence bursts are gray. For each 1-ms time-bin within bursts, the Proximity Ratio (PR; Eq. 1) was calculated (thin semitransparent black lines), and then, PR profiles were smoothened with Chung-Kennedy filtering (thick black lines)109.
