Extended Data Fig. 7. Dependence of the folding equilibrium constant on temperature and structural perturbations observed in the native state on the ribosome.

(A-B) ¹⁹F NMR spectra of FLN5 on and off (Δ6 truncation) the ribosome recorded at a ¹⁹F-Larmor frequency of 470 MHz. Raw spectra are shown in grey, lineshape fits in colour and the total fit in black. Residuals after fitting are shown below each spectrum. (C-D) Nonlinear fits to a modified Gibbs-Helmholtz equation (see methods) of the equilibrium constants on and off the ribosome measured by ¹⁹F NMR (from panels A-B) shown as the mean ± SEM propagated from NMR line shape fits (panel C) and the resulting thermodynamic parameters (mean ± SD from fits, panel D). (E-F) ¹⁹F NMR spectra of the FLN5 mutant E6 on and off the ribosome (Δ2 truncation) recorded at a ¹⁹F-Larmor frequency of 470 MHz. The FLN5Δ2 E6 was chosen due to its suitable stability in this temperature range to quantify both [U] and [N]. Raw spectra are shown in grey, lineshape fits in colour and the total fit in black. Residues after fitting are shown below each spectrum. (G-H) Nonlinear fits to a modified Gibbs-Helmholtz equation (see methods) of the equilibrium constants on and off the ribosome measured by ¹⁹F NMR (from panels E-F) shown as the mean ± SEM propagated from NMR line shape fits (panel G) and the resulting thermodynamic parameters (mean ± SD from fits, panel H). (I) Left: Chemical shift perturbations (CSPs) measured by NMR (¹H-¹³C HMQC) for methyl groups of natively folded FLN5 (RNCs relative to the isolated protein)²⁵. The black datapoints represent the mean ± SD from five different RNC lengths for ease for visualisation. Right: Average CSPs mapped on the crystal structure of FLN5⁹³. (J) CSPs (RNC relative to isolated protein) measured for FLN5 labelled with three different ¹⁹F-tfmF labelling sites by ¹⁹F NMR at linker lengths of 47 and 67 amino acids⁶. (K) Correlation plots (along with Pearson correlation coefficients) of methyl relaxation parameters ($S_{\mathit{axis}}^2$τ_C) for natively folded FLN5²⁵ in different concentrations of glycerol (left panel) and correlating FLN5 on and off the ribosome (right panel).

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