Extended Data Fig. 5. Characterisation of dynamic processes on and off the ribosome by 19F NMR spectroscopy.
a, On-resonance 19F R1ρ measurements of isolated FLN5 using a high spin-lock field (7500 Hz). Inset shows plot of relative signal intensities from fitted spectra as a function of spin-lock time. The R1ρ determined is consistent with R2 measured using lineshape analysis of the 1D 19F NMR spectrum (23.3 ± 0.8 s-1, Fig S3), indicating the absence of chemical exchange processes. b, On-resonance 19F R1ρ measurements of FLN5 + 34 RNC. Due to limitations in sensitivity, we selected three spin-lock times. Observed spectra are shown above. Spectra shown below were globally fitted, with shared chemical shifts and linewidths, but independent signal intensities. c, Signal intensities determined from a global fit of spectra shown in b were plotted against spin-lock times, and compared against the expected signal decay from R2 measurements determined by lineshape analysis of the 1D 19F NMR spectrum as shown in the shaded regions. Error bars indicate errors determined by bootstrapping of residuals from NMR line shape fittings. d, 19F longitudinal relaxation rate (R1) measurements for the unfolded and intermediate states in FLN5∆6 P742A, used in the CEST measurement fittings. Error determined from data fits. e, 19F CEST profiles for FLN5∆6 P742A measuring exchange between the unfolded and isolated intermediate states using different B1 field strengths (30, 60 Hz). Error determined from data fits. f, 19F CEST measurements of FLN5 + 34 RNC. Due to limitations in sensitivity, we selected six frequencies at which to irradiate (of which one was off-resonance from all NMR peaks and shown in red, with remaining irradiation frequencies indicate d by arrows) with the 15-Hz B1 field. The frequencies were chosen to either saturate N/U states and intermediates (-62.2, -61.8, and -62.6 ppm), or only one intermediate state (-61.2 and -63.1 ppm). In the case of the latter, saturation of I1 (that is at -61.2 ppm) did not result in significant perturbation of the I2 state, and vice versa; this result indicates that the I1 and I2 resonances are distinct, in slow exchange, and therefore provides further evidence that four states are populated by FLN5 + 34. Observed spectra (grey) were fitted (black) by analysing in the time domain using the Bloch-McConnell equations. g, Exchange rates between FLN5 + 34 nascent chain states determined by CEST measurements, using an estimated R1 of 1.1 s-1 for all RNC states. Error determined from data fits.