Supplementary Figure 6: FLN5 RNCs in which FLN6 is substituted with a poly(GS) linker.
From: A structural ensemble of a ribosome–nascent chain complex during cotranslational protein folding
(a) Schematic of the poly(GS)-linker RNCs, in which the FLN6 domain is substituted with a poly(GS) sequence. The FLN6 residues that have been substituted in two RNCs analysed by NMR, FLN5+31 and +42 to generate FLN5+31 GS and +42 GS, respectively, are shown in the highlighted box. (b) Anti-His western blot of FLN5+31 GS and 42 GS RNCs in their released and tRNA-bound forms, respectively. (c) The emergence of FLN5 from the exit tunnel in a series of poly(GS)-linker RNCs was monitored by PEGylation of G751C variants of the FLN5 RNCs in a manner similar to that shown in Fig. 4 for the FLN6 domain. (d) Overlay of 1H-15N correlation spectra of FLN5+42 GS RNC and FLN5+42 RNC. The latter was recorded at 950 MHz and with enhanced resolution, however FLN5+42 and +42 GS RNCs have similar intrinsic linewidths. The close overlay suggests shows the similar unfolded conformational preferences between the two nascent chains i.e. FLN5 is unfolded in the FLN5+42 GS RNC. (e) Overlay of 1H-15N correlation spectra FLN5+42 GS RNC when it is intact (dark green) and released (after ˜20 h of NMR acquisition, light green), which shows the appearance of additional glycine resonances arising from the poly(GS) linker in the released nascent chain. (f) Relative intensities of FLN5+42 (upper panel) and FLN5+31 (lower panel) GS RNCs compared to isolated, unfolded FLN5 (the green trace represents a 5-point moving average). 5-point moving average plots of the relative intensities of FLN5+42 (upper panel) and FLN5+31 (lower panel) RNCs compared to isolated unfolded FLN5 are also shown for comparison (pink). The close overlay of the two indicates that altering the linker does not seem to impart significantly on the conformation and/or ribosome interactions of unfolded FLN5. (g) Overlay of 1H-15N correlation spectra of FLN5+42 GS RNC after ~20 h of NMR acquisition and of natively folded FLN5: upon release from the ribosome, resonances corresponding to natively folded FLN5 are observed.
