Fig. 7: In-Cell NMR reveals MRAS^WT and MRAS^Q71L remain GDP bound in cells, in contrast to KRAS^Q61L which becomes GTP loaded.

a Overlay of ¹H/¹³C SOFAST-HMQC spectra from ¹³C-Ileδ1-KRAS^WT (left) and KRAS^Q61L (right). Spectra from KRAS prebound to GDP (black) or GMPPNP/GTP (red) in buffer are in the background, and in HEK 293T cells seven hours after electroporation on top (green). Ile21 and Ile100 serve as reporting peaks for nucleotide-bound status and are emphasized with red and gray arrows for GTP and GDP, respectively, on KRAS^WT spectra and boxed on KRAS^Q61L spectra. Ile84 served as a normalization peak to permit comparisons over multiple experiments. b Overlay of spectra from ¹³C-Ileδ1-MRAS^WT (left) and MRAS^Q71L (right) preloaded with GDP (black) or GMPPNP (red) in solution, or in HEK 293T cells seven hours after electroporation (green). Presumed MRAS Ile31 (corresponding with KRAS Ile21) is marked. Broadening of Ile31 informs on GDP/GTP loading, as does the line shape of peaks within the blue square (zoomed, inset). ‘REF’ peak was for normalization between biological replicates. c Quantitation of GDP- and GTP-bound fractions of KRAS^WT and KRAS^Q61L in cells. Ile84 was used for normalization. Ile24 is also unchanged by nucleotides and is a control. Data are mean and standard deviation from n = 3 independent experiments. Statistical comparison between wild-type and mutant used a 2-way ANOVA (P = 0.0007) with a 1-way ANOVA for in-group comparisons (wild-type P < 0.0001 and Q61L P = 0.004). At right are resonances observed at Ile21 GDP- and GTP chemical shifts (marked with a 1 and 2 on spectra in a). d Quantitation of peak intensity for MRAS^WT (left) and MRAS^Q71L (right) in HEK 293T cells. Loss of the GDP-specific peak reports on GTP loading. ‘REF’ is stable independent of nucleotide and was used for normalization. Control peak is also unchanging. Data are mean and standard deviation from at least n = 3 independent experiments. Statistical analysis between wild-type and mutant used a 2-way ANOVA (P = 0.618) and in-group comparisons used t tests (wild-type P = 0.444 and Q71L P = 0.208). At right are resonances at the Ile31 (GDP) chemical shift (marked with a 1 on spectra in b). Source data are provided as a Source Data file.