Fig. 3: Pre-active and active state populations of β₁AR are agonist-dependent and affect ternary complex formation with mini-G_s.

a Overlay of 2D ¹H-¹³C HMQC spectra of ligand-bound β₁AR-E with the full agonist isoprenaline (blue) or the partial agonists xamoterol (intermediate efficacy) (orange) or salbutamol (high efficacy) (magenta). Assignments of peaks related to the pre-active state are indicated by (^P) while those related to the active state are indicated by (^A). b Affinity of β₁AR (β₁AR-E - black, β₁AR-W – grey) ternary complex formation with mini-G_s in the presence of full (isoprenaline) or partial agonists (xamoterol, salbutamol) measured by BLI. Values are the averages of n = 3 individual repeats, and error bars indicate the SD of these replicates. See Methods and Supplementary Methods for details. c Overlay of 1D ¹⁹F NMR spectra of ^TETC344^7.54 of β₁AR-E ligand-bound to either isoprenaline (dark blue) or xamoterol (orange), or in ternary complex with mini-G_s (8 molar equivalents) and xamoterol (red) or mini-G_s (2 molar equivalents) and isoprenaline (light blue). Differences in the signal positions of the ternary complexes indicate ligand dependent variations in the TM7/IL4 region. Vertical dotted lines indicate the signal position of the ternary complexes. Known degradation products are marked by an asterisk. d, e ¹⁹F NMR signal of ^TETC344^7.54 for β₁AR-E bound to isoprenaline (d) or xamoterol (e) reveals the existence of two receptor populations (active state A and pre-active state P) when bound to isoprenaline, but only one with xamoterol. The recorded spectrum is shown in black, with the deconvoluted signal components shown in green. The reconstructed spectrum is shown in red with the residual trace shown in blue. Peak positions are indicated by vertical dotted lines and labelled according to the active state (A) or the pre-active state (P). The asterisk indicates a known degradation product. Side-by-side comparison of ¹H-¹³C spectra of β₁AR-E-L289M^6.34 in ternary complex with mini-G_s (8 molar equivalents) in the presence of xamoterol (f), salbutamol (g) or isoprenaline (h), respectively. The black arrow heads indicate positions of residual ligand-bound receptor signals that are present due to the reduced affinity of the partial agonist-bound ternary complexes for mini-G_s.