Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

I n the cell, most nascent polypeptide chains begin to fold during biosynthesis [1][2][3] . In many cases, co-translational folding increases the ability of a protein to efficiently attain its native structure [4][5][6][7] . This may in part be due to the ribosome modulating the conformational ensembles sampled by nascent chains [8][9][10][11] . The ribosome constrains disordered chains close to its charged surface 12 , and can promote the early formation of compact states during co-translational folding [13][14][15][16] . In general, nascent chains have been found to be destabilized when they are bound to the ribosome 5,[17][18][19] . This could be linked to interactions between the disordered nascent chain and the ribosome surface 20 , some of which bear a partial electrostatic character 21,22 . Ribosome-nascent chain interactions have also been suggested to attenuate co-translational folding rates 23 , and to compete with co-translational assembly between a nascent chain and its binding partner 24 . Collectively, these studies point clearly towards a role for the ribosome in shaping the onset of co-translational folding 3 . However, due in large part to the technical difficulty of measuring the intramolecular equilibria associated with ribosome-nascent chain interactions, a link between the energetics of ribosome interactions and co-translational folding outcomes has not yet been established in quantitative terms.
In this article we study the co-translational folding of FLN5, a 105 residue immunoglobulin-like domain from the tandem repeat protein filamin (Fig. 1a), using SecM-arrested ribosome-nascent chain complexes (RNCs) in which FLN5 is tethered to the ribosome via variable lengths of the following domain, FLN6 (Fig. 1b) 20,25 . Measurements of the accessibility of a C-terminal cysteine to covalent modification by PEG-maleimide (PEGylation) showed that the entire FLN5 domain emerges beyond the ribosome exit tunnel for linkers comprising at least 31 residues 20 (FLN5+31). However, NMR observations demonstrated that FLN5 remains partially unfolded until the linker extends beyond 42 residues. This offset between the emergence of FLN5 and its folding suggests that the ribosome has a destabilizing effect on co-translational folding, which we speculated could be due to interactions between the unfolded nascent chain and the ribosome surface 20 . We use NMR spectroscopy together with protein engineering, molecular dynamics simulations and PEGylation measurements of nascent chain stability to identify a series of interaction sites on the nascent chain of varying affinities, and their impact on co-translational folding.

Results
Identification of regions of FLN5 RNCs interacting with the ribosome surface. Since the FLN5+31 RNC represents the first point during protein biosynthesis when the entire sequence is available for folding, we have used this biosynthetic snapshot as a starting point to examine how the ribosome might modulate the dynamic properties of a nascent chain. Previously, a comparison of the 2D 1 H, 15 N NMR correlation spectrum of a FLN5+31 RNC against an isolated unfolded variant, Y719E, revealed site-selective line broadening that we interpreted as evidence of such interactions (Fig. 1c,d). From this, we have identified three main regions for investigation here. First, two clusters of aromatic residues, located within the core of the folded domain (Fig. 1a), were identified as displaying substantial broadening; these are referred to here as 'F 3 ' (residues F665-F675) and 'Y 3 ' (residues Y715-Y727) sites (Fig. 1d, green). Second, strong line broadenings were observed in the mildly basic C-terminal region of FLN5 (residues N728-C747, the 'C-terminal segment' (Fig. 1d, red). These observations correlate well with measurements of the interaction between individual amino acids and purified 70S ribosomes, which are strongest for aromatic and basic side chains ( Fig. 1e and Extended Data Fig. 1). Third, resonances of the FLN6 tether are broadened to varying extents across FLN5 RNCs of increasing lengths, which, in part, relates to its occlusion within the ribosomal exit tunnel 20 (Fig. 1d, cyan). and A 3 A 3 , in which the clusters of aromatic residues were mutated to alanine ( Fig. 2a and Extended Data Fig. 2). The close overlay between 1 H, 15 N NMR spectra of the isolated variants and disordered FLN5 indicates they are natively unfolded, as expected given large changes to the hydrophobic core (Extended Data Fig. 2c-f). On the ribosome, the 1 H, 15 N correlation spectra of the corresponding FLN5+31 RNC variants showed substantial increases in cross-peak intensities relative to wild-type (wt) FLN5+31 RNC, broadly localized to the alanine mutation sites ( Fig. 2b and Extended Data Fig. 3). Residues between A665 and G700 within the FLN5+31 A 3 A 3 RNC variant reached a relative intensity close to 1, indicating that the mobility of this segment of the nascent chain is comparable to that of the isolated unfolded protein. These data demonstrate that the aromatic residues mediate a large proportion of the interactions at these sites. By contrast, in all variants the C-terminal segment of FLN5 (N728-C747) remained broadened beyond detection. This suggests additional interactions within this nascent chain segment (Fig. 2b).
To measure the strength of these aromatic interactions, we developed and acquired sensitivity-optimized measurements of the transverse cross-correlated relaxation (CCR) rate (Extended Data Fig. 4). Interactions of nascent chain segments with the ribosome surface will result in transferred relaxation, and thus an increase in the CCR rate, Δη xy , relative to the isolated unfolded state. The increase, Δη xy = p B η bound xy , is proportional to the bound fraction p B , and to the CCR rate of the bound state, η bound xy . Based on the known rotational correlation time (τ C ) of the ribosome 26 , η bound xy is estimated to be ∼7,000 s −1 .
CCR measurements were acquired for FLN5+31 wt, F 3 A 3 , A 3 Y 3 and A 3 A 3 RNCs, and the corresponding isolated proteins ( Fig. 2c and Extended Data Fig. 4). In general, we observed that increased CCR rates were associated with reduced resonance intensities. In the wt RNCs, increases in η xy of ∼15 s −1 were measured around the aromatic F 3 cluster. Within the Y 3 site, residues beyond T714 were broadened beyond detection in wt and A 3 Y 3 RNCs, but examination of flanking residues indicates that η xy probably increases substantially beyond 15 s −1 . Increased relaxation was also observed for residues at the N terminus, which we ascribe to an interaction of the 6xHis tag as previously observed in α-synuclein RNCs 21 .
We observe some evidence of cooperativity between the F 3 and Y 3 clusters, both by comparison of resonance intensities and of CCR rates: the elimination of one cluster leads to a small reduction in the interaction of the neighbouring cluster (Fig. 2b,d). However, in quantitative terms the extent of interaction is weak in all cases. Within the F 3 cluster, observed increases in CCR rates correspond to ribosome-bound nascent chain populations of ∼0.1% (Fig. 2d). While resonances for the Y 3 cluster were strongly broadened in wt and A 3 Y 3 , only allowing for a partial quantitation of CCR rates, the ribosome-bound population between residues 708 and 716 also neared ∼0.1%. Although these estimates assume a rigid bound state, even substantial flexibility that results in an order of magnitude decrease in η bound xy would indicate a bound population of only a few percent. Such weak interactions within these regions are not sufficient to perturb the co-translational folding process.
Interaction of the C-terminal segment with the ribosome surface. We next sought to investigate the interaction of the C-terminal segment (N728-C747) (Fig. 1d). The elimination of the Y 3 cluster in the F 3 A 3 and A 3 A 3 variants resulted in newly observable resonances flanking this segment. Careful inspection revealed that small chemical shift perturbations (CSPs) were observed for these resonances (V717-G725, encompassing part of the Y 3 region), relative to the isolated protein, and that their magnitude increased towards the (unobserved) ribosome-binding segment (Fig. 3a,b and Extended Data Fig. 5). Similarly, CSPs were observed at the C-terminal end of the binding segment between I748 and A751. Focusing on the A 3 A 3 variant, we found that these CSPs were substantially reduced at high ionic strength (Fig. 3c), suggesting that they are associated with an interaction mediated at least in part by an electrostatic contribution.
We then explored the effect of the RNC linker length on the observed CSPs. We hypothesized that a shorter RNC would experience a higher effective ribosome concentration 20 and therefore modulate the extent of binding. Indeed, as the linker length is increased from 26 to 110 amino acids, CSPs were observed to decrease (Fig. 3d) while resonance intensities increased (Extended Data Fig. 5). The CSPs at different lengths were collinear, indicating that changes in the 1 H and 15 N chemical shifts were strongly correlated (Fig. 3d). These collinear, correlated CSPs are an unambiguous indication that the C-terminal segment is rapidly exchanging between a ribosome-bound and free state, such that the observed chemical shift reflects a population-weighted average of unbound and bound states 27 . Resonances of the C-terminal residues I749-A751 showed deviations from collinearity at short linker lengths, which we attribute to proximity to the exit tunnel. For this reason, they have been excluded from further analysis. Together, these observations provide compelling evidence for a strong ribosome interaction involving nascent chain residues between Y727 and C747.
Alanine mutations within the F 3 and Y 3 clusters did not substantially perturb the observed C-terminal CSPs, indicating that there is no detectable cooperativity between interactions of these aromatic clusters and the C-terminal segment (Extended Data Fig. 5). Furthermore, no modulations in chemical shifts or intensities were observed upon varying magnetic field strength (Extended Data Fig. 5). Given the fast chemical exchange behaviour observed, we can infer . Aromatic (green) and surface-exposed (red) residues that have been mutated in this study are shown using stick representations. b, Design of FLN5+L rNC constructs, where L indicates the linker length, comprising a fragment of the following FLN6 domain and the 17-amino-acid SecM arrest peptide 20 . c, 1 H, 15 N SOFAST-HMQC spectrum of a FLN5+31 rNC (blue) overlaid with that of the isolated, unfolded FLN5 Y719e variant (grey) (283 K, 950 MHz). Contour levels of the rNC spectrum are 3.8-fold lower than for isolated Y719e. d, Intensities of FLN5+31 rNC resonances relative to the isolated, unfolded FLN5 Y719e variant. A three-point moving average is shown. Shading indicates the approximate location of ribosome interaction sites characterized in this study. e, Transferred transverse 1 H NMr relaxation measurements of the interaction of free amino acids with purified 70S ribosomes (283 K, 700 MHz). All error bars indicate standard errors derived from the spectral noise.
from the largest frequency difference, Δν, of 30 Hz (for the 15 N resonance of E724 at 22.3 T) that the dissociation of the bound state is rapid, with a lifetime much less than 3 ms (τ ≪ 1/4πΔν, that is,

Impact of the linker sequence composition on FLN5 interactions.
We investigated the influence of the FLN6 linker on FLN5-ribosome interactions. We previously reported that replacing all FLN6 residues with a poly(GS) sequence ( Fig. 3e) did not perturb co-translational folding equilibria 20 . Here we find that substituting poly(GS) linkers within A 3 A 3 RNCs led to only small shifts in the CSPs and resonance intensities in the C-terminal segment, indicating that the extent of binding is not greatly perturbed ( Fig. 3f and Extended Data Fig. 6), and suggesting little impact of the nature of the tether on the co-translational folding of FLN5.
Quantification and molecular modelling of the C-terminal segment interaction. Having identified a ribosome interaction site in the C-terminal segment, we next examined its molecular basis. Although the C-terminal segment does not contain aromatic residues, it contains three basic side chains (R734, K739, K746) that we hypothesized would contribute towards the electrostatic interactions ( Fig. 1e), given the reduced CSPs observed at increased ionic strength (Fig. 3c). To explore this, we designed a FLN5 construct, A 3 A 3 E 6 , in which six residues (surface-exposed within the folded state) were replaced with acidic glutamate residues, thus reversing the net charge within this segment from +1 to −6 (Figs. 1a and 4a). The E 6 mutations were found to greatly reduce the magnitude of CSPs in the C-terminal segment (Fig. 4b), indicating that these mutations reduce the affinity of the segment for the ribosome surface. However, some CSPs and line broadening persist (Extended Data Fig. 7), suggesting that the binding interaction of this region is not abrogated completely. Similar reductions in CSPs were also observed between FLN5+31 wt and E6 RNCs, although due to the increased line broadening fewer resonances adjacent to the interacting segment could be resolved (Extended Data Fig. 7).
To quantify the effect of the E 6 mutations on the binding interaction, we have analysed further the observed CSPs, which reflect a population-weighted average of unbound and bound states. These residues provide a convenient 'ruler' to compare the interactions of different nascent chains with the ribosome. However, to determine the absolute amount of binding, the chemical shift of the fully bound state must be determined. To achieve this, we carried out a global analysis of CSPs observed in the A 3 A 3 and A 3 A 3 E 6 RNC variants across multiple linker lengths (Fig. 4c). Two assumptions were required for this analysis. First, while the strength of interactions in these variants clearly varies as a function of linker length, we assume that the difference in free energy of binding between variants, ΔΔG A3A3−A3A3E6 U free −U bound (where U free and U bound represent unfolded states with the C-terminal segment unbound and ribosome-bound, respectively), is independent of RNC length. This is equivalent to the assumption that C-terminal segments in both variants experience the same effective ribosome concentration at a given linker length, which is supported by the similar structural and dynamic properties of the A 3 A 3 and A 3 A 3 E 6 variants: no chemical shift perturbations or differences in 15 N R 2 relaxation rates are observed beyond the immediate vicinity of the E 6 mutations (Extended Data Fig. 8). Second, while unbound resonance positions vary between A 3 A 3 and A 3 A 3 E 6 RNCs due to local sequence effects, we assume that the chemical shift change upon binding, Δδ max , is the same for both variants. Given this, the observed chemical shift perturbations of four well-resolved resonances in A 3 A 3 and A 3 A 3 E 6 RNCs were fitted to determine the chemical shift differences between free and bound states, and the difference in affinities of the two variants, ΔΔG A3A3−A3A3E6 U free −U bound = 1.9 ± 0.1 kcal mol −1 (Fig. 4d and Extended Data Fig. 8). These results indicate that at a short RNC length of 26 amino acids, 90% and 22% of the A 3 A 3 and A 3 A 3 E 6 RNCs are bound to the ribosome, respectively, then at a longer RNC length of 42 amino acids these values decreased to 60% and 7%, respectively, and by 110 amino acids, the interaction essentially disappears in the A 3 A 3 E 6 variant (Fig. 4e).
Next, we used coarse-grained (CG) molecular dynamics simulations of the A 3 A 3 and A 3 A 3 E 6 RNCs to probe this interaction further, and to explore the location of nascent chain interaction sites on the ribosome surface (Fig. 4g). The strength of electrostatic interactions between the nascent chain and the ribosome surface in the CG model was calibrated using simulations of the A 3 A 3 FLN5+42 RNC, in order to achieve ∼60% binding of the C-terminal segment (730-746), as determined from our NMR observations (Fig. 4e). Simulations of other RNC lengths (+31, +67 and +110), and of the +42 E 6 variant, were then carried out with no further adjustment of parameters. We found that this CG model accurately identified  , and isolated FLN5 A 3 A 3 (grey). errors were derived from the spectral noise for F 3 A 3 , A 3 Y 3 and A 3 A 3 variants, while the mean and standard error from three biological repeats are shown for the wt. Three-point moving averages are shown. d, Bound state populations, p B , of the highlighted nascent chain segments (residues 660-680 and residues 708-716) determined by analysis of transferred cross-correlated relaxation. error bars represent the standard deviation of residues within these segments.
electrostatics-based interactions of the C-terminal segment of the nascent chain that were consistent with the extent of binding measured from our CSP analysis across all lengths (Fig. 4f). The C-terminal binding segment (N728-C747) in the A 3 A 3 FLN5+31 RNC contacted primarily the 23S rRNA region located adjacent to the exit tunnel (mainly helices H24 and H50), and also a loop of the nearby ribosomal protein, uL24 (Fig. 4h). Moreover, for many individual RNA bases in this region, in particular those within helices H24, H47, H50 and H59 (Extended Data Fig. 9), the contact probability correlated strongly with the extent of binding across nascent chain lengths as determined experimentally from our CSP analysis (Fig. 4i). These observations point to a nascent chain interaction site at the ribosome exit vestibule. We note that a similar interaction site is also observed for folded FLN5+47 RNC by cryo electron microscopy 28 , and that helices H24 and H50 are highly structurally conserved across Bacteria and Eukaryotes. It may be that this region on the ribosomal surface has a role in modulating the co-translational folding of FLN5 and perhaps generally for nascent chains.

Modulation of co-translational folding by C-terminal interactions.
In the previous sections, we have developed a detailed description of the interactions formed between unfolded FLN5 nascent chains and the ribosome surface. By stabilizing the unfolded state, such interactions will in general lead to an inhibition of folding (Fig. 5a), and we set out in this section to test this linkage in quantitative terms. Since interactions of the F 3 and Y 3 aromatic clusters are weak, we have focused our analysis on the strong interactions of the C-terminal segment, which we have previously shown is essential for folding of the domain 29 . A simple calculation (detailed in the Supplementary Information) relates the amount of binding within the unfolded state (p B ) to its change in stability: We have used this expression, together with our measurements of interactions in unfolded RNCs of various lengths, to predict the effect on the co-translational folding equilibria of folding-competent FLN5 RNCs in the absence of the destabilizing A 3 A 3 mutations (Fig. 4e, right-hand axis). While this calculation assumes that the A 3 A 3 mutations themselves do not perturb the interaction, we have previously noted that the impact of E 6 mutations on interactions of wt and A 3 A 3 RNCs was similar (Extended Data Fig. 7), and that there was no detectable cooperativity between the aromatic clusters and interactions of the C-terminal segment (Extended Data Fig. 5). On this basis, we predict a destabilization of wt FLN5 RNCs by over 1 kcal mol −1 at short linker lengths (Fig. 4e, right-hand axis). In the absence of other changes in thermodynamic stability, we also predict that the E 6 variant should shift co-translational folding equilibria towards the folded state ( Fig. 5a and Fig. 4e, right-hand axis).
To test the predicted effect of interactions on folding experimentally, we employed a cysteine mass-tagging approach, PEGylation 30 , as a reporter of folding and co-translational folding equilibria. 2D NMR observations indicated that wt and E 6 FLN5 have similar structures both in isolation and as RNCs (Extended Data Fig. 10),  and so the mutations C747V and A721C were introduced to replace the native, C-terminal cysteine with one that is unambiguously emerged beyond the exit tunnel at all linker lengths examined here, and that is protected from modification by PEG-maleimide unless the domain is unfolded (Extended Data Fig. 10). The isolated variants, FLN5 C721 V747 (the 'pseudo-wild-type') and FLN5 C721 V747 E 6 , were reacted with PEG-maleimide in increasing concentrations of urea and the extent of protection was determined by electrophoresis. The resulting measurements fitted closely to a two-state unfolding model and indicated that the E 6 variant was destabilized relative to the wt by ΔΔG E6−wt N−U = 4.0 ± 0.2 kcal mol −1 (where N and U represent native and unfolded states), in excellent agreement with equivalent CD measurements ( Fig. 5b and Extended Data Fig. 10).

Fig. 3 | A ribosome-binding site in a C-terminal segment of FLN5. a, resonance intensities (circles) and CSPs (bars) for the FLN5+31 A 3 A 3 rNC relative to isolated A 3 A 3 . b, A model for the C-terminal segment in an equilibrium between ribosome-bound and unbound states. c,
We next subjected FLN5 and FLN5 E 6 RNCs to PEGylation following the same protocol ( Fig. 5c and Extended Data Fig. 10). As ribosomes dissociate at high concentrations of urea 17 , we restricted our analysis to measurements below 3 M urea, with the exception of FLN5+110 RNC for which data up to 3.75 M were used (Fig. 5c, dashed lines). The stabilities of FLN5+110 wt and E 6 RNCs are indistinguishable from the corresponding isolated proteins, indicating that the difference in thermodynamic stability between isolated FLN5 and FLN5 E 6 is conserved on the ribosome at long linker lengths (ΔΔG E6−wt N−U = 4.0 ± 0.3 kcal mol −1 ). However, at short linker lengths, the difference in stability between FLN5 and FLN5 E 6 was decreased (ΔΔG E6−wt N−U = 2.3 ± 0.2 kcal mol −1 for FLN5+31) (Fig. 5c,d). Consistent with our hypothesis, wt nascent chains therefore become destabilized relative to E 6 under strongly interacting conditions. Based on our earlier measurements of bound populations (Fig. 4e), we can predict the change in stability relative to the isolated domains (Fig. 5d), and these predictions closely match direct observations using PEGylation (Fig. 5d). Therefore, we conclude that the C-terminal segment can indeed modulate co-translational FLN5 folding through the competition between binding and folding.

Conclusion
For many domains, co-translational folding has been reported to be destabilized on the ribosome 5,[17][18][19] . This effect is generally inferred to arise through interactions between emerging nascent chains and the ribosome, which have been observed for a range of different nascent chain sequences 12 Fig. 7) is shown (red line) from which ΔΔG A3A3−A3A3E6 U free −U bound is derived. e, Binding of the FLN5 C-terminal region as a function of linker length. The right-hand axis shows the predicted perturbation to the observed co-translational folding equilibrium in folding-capable rNCs (equation (2)). f, Interaction propensities of unfolded FLN5 nascent chain residues with the ribosome surface based on coarse-grained molecular dynamics simulations. Markers with error bars indicate bound fractions of the C-terminal region (shaded) determined experimentally (Fig. 4e). g, A structural ensemble of the FLN5+67 nascent chain determined through molecular dynamics simulations. The nascent chain is coloured as in a, with the interacting segment highlighted in red. ribosomal rNA and proteins are coloured white and grey, respectively. h, Contact probabilities between ribosome protein or rNA residues and the C-terminal region of the FLN5+31 A 3 A 3 nascent chain, determined through coarse-grained molecular dynamics simulations. i, Correlation across multiple nascent chain lengths between nascent chain-ribosome contact probabilities and the experimentally determined binding between the ribosome and the C-terminal region of FLN5 nascent chains. the energetics of interactions and of co-translational folding has not previously been established. In this article we have therefore systematically examined a series of interactions between unfolded FLN5 nascent chains and the ribosome surface, in order to determine their effect on co-translational folding. While some of these interactions, between aromatic clusters and the ribosome surface, are too weak to perturb the energetics of folding substantially, we have identified a strongly interacting C-terminal segment, with over 90% bound at short linker lengths. The length of the sequestered segment (22 amino acids) is longer than the C-terminal truncation that can be tolerated by isolated FLN5 before it unfolds (12 amino acids) 29 , supporting the crucial role of this segment to enable native structure formation. Importantly, our analysis establishes quantitative agreement between the strength of the observed interactions and the energetics of co-translational folding itself, providing a residue-specific demonstration of the ability of the ribosome surface to directly modulate co-translational folding, effectively acting as a holdase.
A notable consequence of our analysis is that strong interactions are required to appreciably perturb the co-translational folding landscape, for example, a destabilization of 1 kcal mol −1 requires over 80% binding (equation (2)). Such interactions were indeed observed for the C-terminal segment at short linker lengths, but the bound population decreases sharply with increasing linker length, to below 50% at linker lengths beyond 47 amino acids (Fig. 4e). This rapid, short-range effect may provide a mechanism by which the engagement of molecular chaperones with the emerging NC is regulated.
We observe that the molecular determinants of ribosome interactions, that is, positively charged and aromatic residues, are similar to those recognized by other molecule chaperones and processing complexes 31 . These include signal recognition particle 32 , the ribosome-associated chaperone trigger factor [33][34][35] , and SecB 36 , both of which function as holdases for nascent polypeptide chains, and other downstream chaperones such as DnaK 37 . Indeed, over the past few years NMR studies have been instrumental in revealing with exquisite detail the mechanisms underlying substrate recognition and chaperone action by these molecules 35,36,[38][39][40][41][42] .
During biosynthesis, substrates emerge from the ribosome in their high-energy unfolded states, and so in contrast to chaperones that act post-translationally, interactions with holdases carry no energetic cost. This is reflected in the ATP independence of both trigger factor and SecB holdases-as well as in interactions with the ribosome surface itself. We speculate that this short-range holdase activity could have a number of functional roles: sequestration of hydrophobic segments until later residues have been synthesized; to reduce the risk of misfolding, particularly in tandem repeat proteins such as FLN 29,43 ; or to delay folding ahead of co-translational assembly [44][45][46] or the engagement of downstream chaperones such as TF and SecB, which may be involved in secretory pathways 35,36 . This suggests that the ribosome is more than an inert hub that orchestrates interactions of auxiliary factors and chaperones 47 , and in fact is itself an active participant in the co-translational folding process.
In conclusion, we demonstrate the holdase effect of the ribosome as a bespoke form of regulation over co-translational folding.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41557-021-00796-x. Measurements at low urea concentrations (filled circles) were fitted to a two-state unfolding curve using the m value determined from the isolated proteins. d, Difference in free energy of co-translational folding between wt and e 6 rNCs, as measured by PeGylation (green) and predicted from CSP analysis (orange). errors bars represent the standard error derived from fitting.

Methods
Sample preparation and quality control. DNA constructs of tandem immunoglobulin domains FLN5 and FLN6, and of poly(GS) glycine serine repeat sequence variants and Y719E variants, were described previously 20 13 C HMQC spectra were acquired at 800 MHz with acquisition times of 100 ms and 7 ms in the direct and indirect dimensions, respectively. Sample integrity was assessed through 13 C-edited 1 H STE diffusion measurements 25 and 1 H R 2 relaxation measurements ( 13 C-edited and incorporating a filter to select slow relaxing coherences 55 ), in which signals detected after a 100 ms relaxation delay were interpreted as indicating nascent chain release. 1 H R 2 rates of isolated amino acids (200 µM in D 2 O) were measured in the presence and absence of 1 µM 70S ribosomes, using a 500 Hz PROJECT pulse train (700 MHz, 283 K) 56 .
CD spectroscopy. CD spectra were acquired using a Chirascan-plus CD spectrometer (Applied Photophysics). All samples (20 µM) were incubated for ≥3 h at 283 K prior to measurement in Tico buffer (12 mM Hepes, 30 mM NH 4 Cl, 6 mM MgCl 2 , pH 7.5). CD signals at 211 nm were fitted globally using MatLab to a two-state unfolding model with a common m value, 〈m D-N 〉 (ref. 57 ): where [D] is the denaturant concentration, [D] 50% is the midpoint of folding, and α terms represent baselines for native (N) and denatured states. Stabilities were then calculated as ΔG D-N = m D-N [D] 50% .

Molecular dynamics.
Simulations of FLN5+31, +42, +67 and +110 RNCs were run in Gromacs 4.5.7 58 using Cα structure-based models generated with SMOG 2.0 59,60 , extended to represent RNA using three beads located at P, C4′ and N3 atoms. A rigid ribosome model was created based on the structure 4ybb 61 and including only nascent chain-accessible residues, that is, the exit tunnel and the surrounding surface. The A 3 A 3 variant was modelled by removing all contacts involving the mutated sites, and the simulation temperature was then tuned so that isolated wt and A 3 A 3 variants were folded and unfolded, respectively. Electrostatic interactions were introduced using Debye-Hückel theory 62 , with parameters chosen to reproduce the experimentally observed bound population of the FLN5+42 RNC and then applied without change to the remaining lengths. Three independent simulations were carried out for each system, with ∼2 × 10 8 steps per run.
PEGylation. Samples (100-200 pmol isolated proteins or 8 pmol of RNC) were incubated in increasing urea concentrations for at least 2 h at 283 K prior to measurements. Methoxypolyethylene glycol maleimide (PEG-Mal) was added to a final concentration of 2 mM and incubated for 10 min before quenching the reaction with 0.23 M DTT. Samples were separated on denaturing 12% (w/v) polyacrylamide Bis-Tris gels (pH 5.7) which were subsequently Coomassie stained (for isolated proteins) or analysed by Western blot (for RNCs). Quantitation was performed using ImageStudio (Licor). As a control, Western blots of dilutions of a FLN5+47 RNC variant showed excellent linearity (R 2 = 0.98) over the concentration range used in analyses (Supplementary Fig. 14). Populations of unfolded (PEGylated) nascent chains were determined relative to the total nascent chain concentration (PEGylated and unPEGylated) and fitted to equation (2) to determine the thermodynamic stability as described above.

Data availability
Data supporting the findings of this study are included in the article, source data and Supplementary Information files. Assignments have been deposited in the BMRB under accession codes 51023 and 51028. Source data are provided with this paper.

Code availability
NMR pulse sequences are available on https://github.com/chriswaudby/pp and also as Supplementary Information. Source data are provided with this paper. . (e) Coarse-grained ribosome structure highlighting the location of rNA residues A1336, C490 and A1535, and (f) correlation plots between simulated and experimentally determined nascent chain-ribosome interactions for these residues. Data are shown for linker lengths +31, +42, +67 and +110, and best fit lines through the origin are plotted with correlation coefficients as indicated.