Figure 2.

Elevated rates of basal nucleotide exchange in Gα_i1 mutants. (a) Basal Exchange. The basal rate of GTP-γS binding was determined by monitoring the relative increase in the intrinsic Trp²¹¹ fluorescence. Basal exchange for the wild-type is shown with a solid line (bottom trace), basal exchange for the I56C/Q333C double mutant is shown with a small-dashed line (------), and basal exchange for the D328R mutant is shown with a large-dashed line (——). (b) Receptor catalyzed nucleotide exchange. Nucleotide exchange for the receptor-catalyzed wild-type and mutant Gα_i1 subunits was determined by monitoring the relative increase in the intrinsic Trp²¹¹ fluorescence performed in presence of 500 nM rhodopsin. The addition of rhodopsin does not significantly affect the rate of nucleotide exchange in the I56C/Q333C or D328R Gα_i1 subunits but catalyzes a 4-fold rate enhancement for wild-type Gα_i1 subunits (p=0.0005). (c) Quantitation of the initial rates of basal and rhodopsin-catalyzed nucleotide exchange monitored by the relative increase in the intrinsic Trp²¹¹ fluorescence for wild-type and mutant Gα_i1 subunits. (d) Basal nucleotide exchange of GDP- or GDP-AlF₄⁻-bound Gα_i1 subunits as measured by the increase in emission from BODIPY-labeled GTPγS upon addition of GDP-bound Gα_i1 or GDP-AlF₄⁻-bound Gα_i1 subunits. Relative increase was plotted as percentage of basal BODIPY-GTPγS fluorescence prior to addition of Gα subunits. Data are the average of three independent experiments (**p = 0.0027 and p=0.0001).