Extended Data Fig. 8. Expansion and entropic destabilisation of the unfolded state on the ribosome persist at longer NC linker lengths.

(A-C) PRE-NMR analysis of FLN5 A₃A₃ (labelled at C740, black star) in isolation and at three different RNC linker lengths (FLN5 + 31, FLN5 + 47, FLN5 + 67). Panel A shows a window average over three residues for ease of visualisation. Panels B and C show all datapoints as the fitted mean ± RMSE propagated from spectral noise. The colour scheme in panels B-C is the same as in panel A. Theoretical reference profiles expected for a fully extended polypeptide are also shown as dashed lines. The shaded region at the C-terminus represents the region of FLN5 that is broadening beyond detection through ribosome interactions (N730-K746, in the RNC)⁷. (D-E) ¹⁹F NMR spectra of FLN5 (F672A) on and off the ribosome recorded at a ¹⁹F-Larmor frequency of 470 MHz. A destabilising variant (F672A) is used to enable measurements of the unfolded state populations at FLN5 + 67. Raw spectra are shown in grey, lineshape fits in colour and the total fit in black. Residuals after fitting are shown below each spectrum. (F) Nonlinear fit to a modified Gibbs-Helmholtz equation of the equilibrium constants on and off the ribosome measured by ¹⁹F NMR (mean ± SEM propagated from NMR line shape fits). (G) Thermodynamic parameters estimated from the nonlinear fits in panel F (mean ± SD). FLN5 F672A and FLN5 Δ6 have indistinguishable thermodynamics, validating 672A as a pseudo wild-type system. (H) Nonlinear fit to a modified Gibbs-Helmholtz equation of the equilibrium constants (all constants relative to the unfolded state) on and off the ribosome measured by ¹⁹F NMR (mean ± SEM propagated from NMR line shape fits). (I) Thermodynamic parameters estimated from the nonlinear fits in panel H (mean ± SD). (J) Transverse relaxation rate (R₂) measurements of isolated full-length (FL) FLN5 labelled at position 655 with tfmF recorded at a ¹⁹F-Larmor frequency of 470 MHz and 298 K. (K) 1D ¹⁹F NMR spectra of isolated, full-length FLN5 in different concentrations of glycerol, fitted spectra in blue, raw spectra in grey. (L) Fitting of R₂ rates for FL-FLN5 in different concentrations of glycerol. (M) Correlation between measured R₂ rates (panel L) and those obtained from the linewidths of the peaks in the 1D spectra (panel K). Points are shown as the mean ± SEM propagated from NMR line shape fits. (N) Correlation between the ¹⁹F linewidth/R₂ rate obtained from line shape fitting (mean ± SEM) and previously determined rotational correlation times of FLN5 in different concentrations of glycerol²⁵. (O) 1D ¹⁹F NMR spectrum of FLN5 + 47 used in panel (P). (P) Estimated populations of coTF intermediates I1 and I2 bound to the ribosome based on the experimental ¹⁹F linewidth at 298 K⁶ and linear correlation between linewidth and rotational correlation time (panel N). The ribosome-bound populations were estimated with an $S_{\mathit{bound}}^2=1.0$ (τ_R,bound = 3003 ns) and are shown as the mean ± SEM propagated from fitted NMR linewidths.

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