FIGURE 6.

Comparison of the nucleotide binding properties of Gαs D173N and Gαo D151N. A and C, mutation of Asp-173 to N in His-Gαs dramatically impairs GTPγS binding (A) whereas mutation of Asp-151 to Asn in His-Gαo accelerates it (C). GTPγS binding by Gαs (A) or Gαo (C) WT (black traces) or D→N mutant (red traces) was determined by intrinsic fluorescence measurements as described in “Experimental Procedures.” For Gαo proteins, data are expressed as % of their own maximal binding, which was F/Fo = 1.19 and F/Fo = 1.24 for WT and D151N, respectively. One experiment representative of three is shown. B and D, D173N dramatically impairs Gαs protection from trypsin hydrolysis upon GTPγS or GDP/AlF₄⁻ incubation whereas D151N has no effect on Gαo. His-Gαs WT or R265H (B) and His-Gαo WT or R243H (D) were incubated in the presence of 30 μm GDP or 30 μm GTPγS (top) or 30 μm GDP plus AlCl₃/NaF (AlF₄⁻) (bottom) before treatment with trypsin as described in “Experimental Procedures.” Gels from representative experiment are shown along with graphs displaying the mean ± S.E. of three independent experiments.