Fig. 4: Relaxation dispersion analyses of the StA_SL domain.

a Temperature dependencies of the ¹H8-¹³C8 aromatic TROSY signals of A771 (A_SL domain) and G777 (St domain). Spectra acquired at 10, 15, 20, 25, 30, and 35 °C at the ¹H frequency of 1.0 GHz are shown in brown, violet, orange, red, green, and blue, respectively. b Relaxation dispersion curve fitting. R_2,eff values obtained at 30 °C and at the ¹³C frequencies of 250 MHz and 150 MHz (¹H frequencies of 1.0 GHz and 600 MHz) are shown by red and blue points, respectively. The relaxation dispersion profile of G777 was fit with the 3-site exchange model, in which it possesses two different chemical shifts in the excited states, using kinetic parameters obtained from the global fitting with the 2-site exchange model (see also Supplementary Fig. 8). The fitted curves are shown as red and blue lines for the ¹³C frequencies of 250 MHz and 150 MHz, respectively. Error bars indicate experimental errors derived from the signal-to-noise ratio of each correlation, as written in Methods. Source data are provided as a Source Data file. c Top: The exchange parameters obtained from the relaxation dispersion profile at 30 °C. Bottom: The chemical shifts of ¹³C8 of G777. Among the observed chemical shifts, 137.9 ppm is the kinetic average of the ground state (138.2 ppm), excited state 1 (136.6 ppm), and excited state 2 (138.5 ppm). Predicted chemical shift values of ¹³C8 of G777 from the ideal A-form helix structure (137.0 ppm), the NMR structure of J-K-St in the free-state (138.1 ppm), and the cryo-EM structure of J-K-St in complex with eIF4G^HEAT1 (140.6 ppm) are shown in gray, indicating that the ¹³C8 chemical shifts mainly reflect the stacking with the preceding residue. Note that the predicted chemical shift values are corrected for the TROSY signals by adding 0.5 ppm.