Fig. 5.

ΔFRET measurements from v-β2AR-Spep sensors correlate with molecular efficacy and β2AR-Spep interaction energy in the fully coupled orientation. (A) List of the adrenoreceptor ligands presented in B–G. (B) Change in ΔFRET for v-β2AR-Spep sensors compared to previously reported GTP turnover for purified recombinant β2AR combined with Gs (9). LS is in black (see Materials and Methods). HBS buffer is in gray (see Materials and Methods). (C) ΔFRET for v-β2AR-Spep sensors compared to previously documented molecular efficacies of indicated ligands (9). Ligand-induced ΔFRET correlates strongly with molecular efficacy and GTP turnover (R² values indicated). (D) Fully active state structures of β2AR (Protein Data Bank [PDB]: 3SN6) (25) and AA_2AR (PDB: 5G53) (34) are shown with receptor in gray surface representation (β2AR) and Gα-α5 helices shown as ribbons (residues 369 to 394 in maroon and residues 368 to 394 in pink). Hot spot residues H387, Y391, and E392 are shown as lines and R389 as balls and sticks. In the AA_2AR structure, only Cα and Cβ atoms of residue R389 could be resolved and no atoms from D368. Relative rmsd between peptide orientations in two structures was 2.0 Å. (E) The intermediate state structure of β2AR and Gαs (PDB: 6E67) (11) is shown with crystallized Gαs peptide residues 381 to 394 as a green ribbon. (F and G) Intermolecular β2AR-Spep interaction energy was calculated over S-peptide residues 368 to 394 based on MD simulations of the fully coupled (F) and intermediate (G) states. MD data points were derived from the last 100 ns of five or eight replicates (as noted in SI Appendix, Table S3). ΔFRET data are derived from at least three independent experiments (at least three separate GPMV preparations). Error bar denotes SEM.