Fig. 1: G peptide-receptor interactions show two distinct kinetic profiles.

a Schematic showing the FRET construct and ΔFRET assay. Nb6B9 binds free receptor (HR’ and HR*) to quench the agonist activated β2AR interaction with Spep in β2AR-Spep SPASM sensors in native membranes prepared from Hek293T cells. b Change in FRET ratio (mCit/mCer) with isoproterenol treatment (iso, 100 μM) with and without of 10 μM Nb6B9. Nb6B9 decreases the Spep activation. Data are presented as mean +/- the standard deviation from 4 membrane preparations (n = 4). c Schematic of the stopped-flow injection set-up used to measure decay in the FRET ratio of agonist activated β2AR-Spep SPASM sensors after mixing with 10 μM Nb6B9. Both syringes contain 100 μM isoproterenol. d Representative decay curve of the decrease in FRET ratio over time when syringe 1 contains 100 μM iso (grey) or 100 μM iso + 10 μM Nb6B9 (purple). The iso curve (grey) was fit a single exponential decay with the rate k_ns. The Nb6B9 decay (purple) was fit to a triple exponential decay, constraining one decay to the iso fit (see Supplementary Fig 7 for all decay curves and fitting parameters). e Lifetime (1/k_{off G}) of two states of the Nb6B9 decay and relative proportion of the slow off and fast off states. Data are presented as mean +/− standard deviation from 4 membrane preparations (n = 4). f Kinetic model with constants constrained by experimentally measured rates. g Simulated Nb6B9 quench data show the interconversion rates from HR’G → HR*G must have lifetimes lower than the duration of the decay (see Supplementary Figure 8 for details of modeling). Source data are provided in the Source Data File.