Fig. 2: ¹⁹F NMR spectroscopy of β₁AR TM6 and TM7.

The individual helices were studied by monitoring A282C^TET (a) and ^TETC344 (b), respectively, highlighting the response of the receptor to agonist binding and the formation of the ternary complex coupled to Nb6B9. All spectra were obtained at 308 K, 564 MHz (¹⁹F) with receptor concentrations of 20 to 30 µM and saturating ligand concentrations (1 mM). a ¹⁹F NMR spectra are shown for A282C^TET for apo β₁AR (light green), bound to full agonist isoprenaline (red) and with isoprenaline in ternary complex coupled to Nb6B9 (black). The peak corresponding to TM6 A282C^TET in the apo and isoprenaline bound β₁AR (P1) appears at a chemical shift of −66.3 ppm. Addition of a two-fold molar excess of Nb6B9 causes a downfield shift of 0.8 ppm to −65.5 ppm (P4). The line marked with Δ indicates the appearance of free TET due to the slow cleavage of the S-S bond at 308 K. b ¹⁹F NMR spectra for TM7 C344^TET show a peak at −65.4 ppm (P2) for the apo β₁AR (light green). Isoprenaline binding (red) causes an upfield shift by 0.06 ppm and a doubling of the linewidth. Coupling to Nb6B9 (black) shifts the signal of the ternary complex (P5) downfield to −64.8 ppm, together with a dramatic increase in the linewidth, compared to both isoprenaline bound and apo β₁AR. For ^TETC344 on TM7 the chemical shifts (c) and the linewidths (d) of P2 correlate with the G_s efficacy of the agonists tested. Chemical shifts and linewidths are shown relative to the apo receptor (δ = −54.42 ppm, Δv_1/2 = 46 Hz). Linear fits of the correlations are indicated by a line and R² values are given (ATE atenolol, CVD carvedilol, ALP alprenolol, CYA cyanopindolol, XAM xamoterol, ISO isoprenaline, ADR adrenaline). e Fast timescale μs-to-ms conformational dynamics of P2 assessed by ¹⁹F CPMG relaxation dispersion measurements for isoprenaline (red) (studied at ¹⁹F frequencies of 564 MHz and 658 MHz) and xamoterol bound receptor (orange) (¹⁹F 564 MHz). Best fit curves to the dispersion data are shown together with values for k_ex and p_P3 (p_I2) obtained from the fits. The apo form of β₁AR does not show any relaxation dispersion (green).