FIGURE 2.

cmp4 counteracts nucleotide dissociation from Ras complex. (A) Scheme depicting the different experiments for nucleotide dissociation or exchange, using nucleotides (GDP or GTP, namely GXP) conjugated with the fluorescent moiety MANT; (B,C) inhibitory efficacy of cmp4 on GEF-mediated nucleotide dissociation (B,C) exchange on HRas (black) and HRas^G13D (red). (D) Mass spectrometry analysis of HRas-GDP in presence of cmp4. The dashed peaks correspond to the expected positions of the nucleotide free-Ras and Ras complexed with cmp4 without GDP. (E,F) Inhibitory efficacy of cmp4 on intrinsic nucleotide dissociation (E) and exchange (F) on HRas^G13D; the initial dissociation or exchange rate of each reaction was determined computing the first derivative at time 0 of the fitted curves reported in Supplementary Figure S4. (G,H) EDTA-mediated competition between mant-GDP loaded on H-Ras and free unlabelled GDP (G) or GTP (H).