Figure 6.

Purification, FCS and solution X-ray scattering studies of Gsα and α^chi. Final purification of Gsα (A) and α^chi (B) with a Superdex 200 column. The inset shows an SDS gel, which corresponds to the shaded area (gray) of the elution peak, pooled, and applied on the gel. Lanes 1 and 2 reveal the purified protein and protein markers, respectively. Binding properties of subunits Gsα and α^chi to fluorescently labeled nucleotides. C and D, results of FCS experiments, showing the binding of labeled nucleotides to subunit α. The upper left and lower right insets show the normalized autocorrelation curves of MgATP- (C) and MgADP-ATTO-647N (D) obtained by increasing the quantity of subunits Gsα and α^chi (increased protein concentration from left to right). C, binding of subunits Gsα and α^chi to MgATP-ATTO-647N and D, MgADP-ATTO-647N displayed as relative bound fraction versus protein concentration. The best fits to titration curves are shown as a non-linear, logistic curve fits. The percentage of complex formation for each concentration was calculated using a two-component fitting model. The binding constant, K_D, was derived by fitting the data with the Hill equation. E, small angle X-ray scattering pattern (○) for Gsα (green) and α^chi (orange). Fitting of the theoretical scattering curve (—) for Gsα computed by CRYSOL with the experimental scattering pattern (○) for Gsα (green) and α^chi (orange) resulted in χ²-values of 1.44 and 1.46, respectively. The curves are displayed in logarithmic units for clarity. Inset, Guinier plots show linearity indicating no aggregation. F, pair-distance distribution function P(r) for Gsα (green) and α^chi (orange). Inset, normalized Kratky plot indicating the folded nature of the protein. G, the average solution shape of Gsα (green) and α^chi (wheat) as calculated by the DAMMIN program is overlapped with the crystal structure of Gsα (brown).