Fig. 4
Assessment of nucleotide binding by p190RhoGAP pG1. a–c Fluorescence of MANT-GTPγS monitored over time upon addition of proteins as indicated. a pG1 from 190RhoGAP-A or -B, or Rac1 as a control, in the presence of 20 mM EDTA plus 10 mM MgCl2. b p190RhoGAP-A pG1 of Xenopus laevis (frog), Gallus gallus (chicken) and Danio rerio (zebrafish) or the single p190RhoGAP of Drosophila melanogaster. c p190RhoGAP-A pG1 in the presence of different divalent cations. Rac1 in Mg2+ is included as a positive control. Uncorrected for photobleaching. d–i Thermal shift assay. d–f Thermal denaturation curves (from a representative experiment) of pG1 domains or Rac1 in the presence of divalent cation, nucleotide or both as listed in f. Buffer alone curves (black) are labeled with arrows. g–i Histograms of melting temperature changes (ΔT m) compared to buffer alone, determined by fitting the thermal denaturation curves (d–f) to a sigmoidal model. Error bars indicate s.e.m. (n=3). Positive shifts indicate a stabilization in the presence of ligand
