Figure 6.

Glutathionylated KRAS^G12Cshows altered biochemical function.A, relative ABD-F reactivity of KRAS^G12C/C118S pretreated with GSSG for the indicated times at a 1:10 ratio. Following pretreatment, KRAS (10 μM) was buffer exchanged and reacted with ABD-F (1 mM) at pH 8.0. No ABD-F reactivity as monitored by fluorescence increase is seen with KRAS^G12C/C118S after 1 h pretreatment with GSSG indicating complete modification of the reactive cysteine by GSSG. Error bars, mean ± s.e.m. B, intrinsic nucleotide dissociation rates of WT, mutant, and glutathionylated KRAS^G12C. Nucleotide dissociation was measured by monitoring the decrease in ^mantGDP fluorescence over time after addition of unlabeled GDP. Data shown are averaged from three or more independent experiments. ∗∗∗p ≤ 0.001, by one-way ANOVA. Error bars, mean ± s.e.m. C, GEF-mediated nucleotide dissociation rates of WT, mutant, and glutathionylated KRAS^G12C measured as in panel B but in the presence of the catalytic domain of the RAS GEF SOS1 (SOS^cat) at a 1:2 ratio. Data shown are averaged from three or more independent experiments. ∗∗∗p ≤ 0.001; ∗∗p ≤ 0.01, by one-way ANOVA. Error bars, mean ± s.e.m. D, dissociation constants (K_d) of WT, mutant, and glutathionylated KRAS^G12C in complex with BRAF-RBD as determined by inhibition of nucleotide dissociation using a nonhydrolyzable analog of fluorescent GTP, ^mantGMPPNP. Data shown are averaged from three or more independent experiments. Error bars, mean ± s.e.m. E, CD melting temperatures of KRAS^G12C modified by CysSNO (nitrosation) and GSSG for 60 min before analysis. Data shown are representative from three or more independent experiments. ABD-F, 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole; CD, circular dichroism; GEF, guanine nucleotide exchange factor; SOS1, Son-of-Sevenless; RBD, Ras-binding domain.