Figure 4.

Experimental validation of spontaneous Gα domain separation in GDP-bound heterotrimeric G proteins and its role in nucleotide exchange. (A) DEER distance distributions measured between spin labels attached to the Ras and helical domains of G_i (Glu238 and Arg90) and G_s (Asn261 and Asn112) show multiple distance peaks, consistent with an equilibrium between closed and open conformations of the α subunit in the presence of GDP, despite the absence of an activated receptor. These distance distributions extend to much larger values than would be expected if the G proteins maintained their crystallographic nucleotide-bound conformations (Fig. S15). (B) Domain separation impacts the basal GDP release rate. The G_i-HD-tether construct (Fig. S16), designed to restrict domain separation, exchanges nucleotides 20-fold more slowly than G_i wild type, under conditions where GDP release is rate-limiting. GDP release was monitored by BODIPY-GTPγS binding kinetics, shown for G_i wild type (black) and G_i-HD-tether (purple). The inset corresponds to the gray dashed box.