Extended Data Fig. 4 |. Characterization of the binding kinetics between NTS-NTSR1 and G_i in cNDs.

a-b, Fitting of Bio-Layer Interferometry (BLI) traces of G_i binding to NTS-NTSR1-cND using (a) one binding mode and (b) two binding mode shows better fitting using two binding mode. Right, a table showing k_on, k_off and K_D from the two binding mode fitting. c, Dissociation between G_i and NTS-NTSR1-cND in the absence (black and brown) and presence (green and blue) of GTPγS, showing faster dissociation of the complex in the presence of GTPγS, suggesting that the NTSR1-Gα_i1β₁γ₁ complex in cNDs is capable of GDP/GTP exchange. d, Association and dissociation kinetics of G_i binding to NTS-NTSR1-cND (dark) and empty cND (gray), showing much slower association and faster dissociation of G_i binding to empty cND compared to NTS-NTSR1-cND, suggesting that interaction between G_i and NTS-NTSR1-cND is driven by G_i binding to NTSR1 rather than to the nanodisc. e, Microscale thermophoresis (MST) data for the binding between NTSR1 and G_i (square mark), as well as the binding between mutant TM86V-L167R E166A/K176A/K178A/S182A/R185A and G_i (triangle mark) in POPC/POPG (3/2) cND. f, MST data for the binding between NTSR1 and G_i in POPC cND (triangle mark), POPG cND (diamond mark) and POPC/POPG/CHS cND (square mark). Right, a table showing K_D from e-f.