Figure 4. Folding of FLN5 on the ribosome monitored by NMR spectroscopy and PEGylation.
(a) FLN5 nascent chain folding as measured by intensity changes of 15N amide resonances (blue) arising from the unfolded FLN5 domain (mean ± s.d. for n=4 (n=3 for +45 and n=2 +47); nascent chain concentration from western blot replicates), and intensity changes in Ile 13CH3 resonances (cyan) arising from native FLN5 structure (mean ± s.d. of spectral noise, n=1). Intensities are normalized and scaled relative to L = 21 (unfolded) or L = 110 (folded). The solvent accessibility of the FLN5 domain from the ribosomal exit tunnel was probed using PEGylation (orange) (mean ± s.d) of folding-incompetent FLN5 Y719E RNCs, where the native Cys747 is close to the FLN5 and FLN6 boundary. (b) Cys747 PEGylation of FLN5 Y719E RNCs results in a band shift (PEG-RNC). (c) C-terminal truncations of isolated FLN5 as measured by NMR. Averaged cross-peak intensities of folded (black) and unfolded (grey) states of FLN5 (see Supplementary Fig. 5) are mapped against truncation length.