Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel

Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel Meranda M. Masse, Valeria Guzman-Luna, Angela E. Varela, Rachel B. Hutchinson, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2734168/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 May, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, no direct information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp 1 − 140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp 1 − 189 ) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface. Biological sciences/Biophysics/Biopolymers in vivo Biological sciences/Cell biology/Protein folding Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Chemical biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Recent evidence suggests that the ribosome plays an active role in cotranslational protein folding and solubility 1 – 8 . During translation, the nascent chain traverses the ribosomal exit tunnel, which is ca. 80–100 Å long, 10–35 Å wide 9 – 12 and typically fits 30 to 40 nascent residues. 13 – 18 Within the ribosomal exit tunnel and its nearby regions across the highly negatively charged outer surface of the ribosome 19 , nascent chains encoding single-domain proteins become compact 20 – 22 and acquire some secondary 23 – 27 and tertiary structure 5,28−32 . This set of observations proves the importance of the ribosome in nascent-protein structure formation. During translation, the ribosome influences nascent protein chains at different levels. For instance, it renders nascent chains soluble relative to the corresponding ribosome-released proteins, thereby supporting cotranslational events devoid of undesirable aggregation 7 . Further, the inner geometry of the ribosomal exit tunnel favors formation of secondary nascent-chain structure, especially of α-helical 26,33−35 or β-sheet nature 27 . In addition, the ribosomal exit tunnel and vestibule enable acceleration of folding – but not unfolding – of a small single-domain protein, thereby stabilizing nascent protein chains relative to their free state in solution 36 . This effect was ascribed primarily to electrostatic interactions between nascent proteins and ribosome 36 . On the other hand, the ribosome may also destabilize single-protein domains, in case the domain is far removed from the peptidyl transferase center 37 . Collectively, these results highlight the influence of the ribosome on nascent protein folding. The ribosome is also known to establish physical noncovalent interactions with some nascent chains. As summarized in Table S1 , these interactions were identified in a variety of experimental studies and can be divided into three categories. Namely, (i) interactions between the ribosome and nascent chains carrying an N-terminal signal sequence 18,38−42 , (ii) interactions between the ribosome and nascent chains bearing a C-terminal ribosome-stalling or arrest sequence 43 – 50 , and (iii) interactions between the ribosome and nascent chains encoding intrinsically disordered proteins 51 . Additional studies are consistent with the presence of ribosome-nascent-chain interactions, though they do not directly prove their existence 52 – 55 . In summary, ribosome-bound nascent chains (RNCs) of intrinsically disordered character or bearing signal or arrest sequences are known to interact with ribosomal proteins. However, RNCs bearing foldable protein sequences and lacking linkers or signal/arrest tags have not been explicitly characterized in terms of experimentally detectable interactions with the ribosome. Further, little is known about how the nascent chain may affect certain components of the ribosome. For instance, empty 70S ribosomes are known to be more prone to dissociation than ribosomes bearing both mRNA and peptidyl tRNA. This conclusion was reached upon addition of either ribosome-dissociation factors 56 or Hofmeister cosolutes 57 – 59 . Other researchers established a similar finding upon depletion of magnesium ions 60 – 62 . In a different study, addition of Hofmeister salts were employed to show that translation initiation complexes (including 70S in complex with initiator tRNA) disassemble more easily than peptidyl tRNAs bearing nascent chains 63 . Ribosomes carrying longer nascent chains were found to be less prone to dissociation 63 . Other studies examined the effect of magnesium ions and other Hofmeister ions on empty-70S-ribosome disassembly and how it changes sedimentation coefficients 64 . Yet, there is only a limited number of studies targeting the effect of non-Hofmeister denaturing agents on the ribosome. For instance, it is known that the 30S subunit disassembles in the presence of 6 M urea 65 . In addition, urea lowers the melting temperature and sedimentation coefficient of the 50S ribosomal subunit 66 . The 30S subunit is more sensitive to thermal denaturation than the 50S subunit, and the 70S ribosome is most thermally stable 67 . In addition, 70S ribosomes bearing a nascent chain are less prone to chemical denaturation than empty ribosomes 37 . Here, we address the lack of knowledge on nascent-chain/ribosome interactions by exploring them in the case of ribosome-bound nascent chains (RNCs) of increasing length belonging to a foldable protein sequence. We find that RNCs up to chain length 140 interact only with one ribosomal protein (r-protein), i.e., L23, in the vicinity of the ribosomal exit tunnel. This result is surprising because the ribosomal surface near the tunnel exit bears several r-proteins. A wider interaction network, including one additional ribosomal protein and the trigger factor (TF) chaperone, gets established as the nascent chain elongates up to 189 residues. The populations of RNC-interacting proteins evolve as a function of chain elongation. Specifically, interactions with r-proteins get partially or completely replaced by interactions with the TF molecular chaperone, as TF concentration increases up to physiologically relevant values. In order to gain additional insights on the potential stabilizing role of the interactions, we also investigate the effect of the above RNC/r-protein interactions on the bacterial ribosome. The apparent stability of the complexes between RNCs of foldable and intrinsically disordered protein sequences and specific r-proteins is weak and, surprisingly, does not vary significantly with RNC sequence, length, net charge and hydrophobicity. Hence, we propose that the ribosome provides unbiased thermodynamic assistance to nascent chains regardless of their electrostatic and nonpolar character. As an ancillary finding, we also show that the apparent thermodynamic stability of the peptidyl transferase center (PTC) and all ribosomal proteins is not affected by RNC-ribosome interactions. Further, short peptidyl-tRNAs (snc-tRNAs) stabilize the 70S ribosome against denaturation by the non-Hofmeister cosolute urea, suggesting a multi-step model for the disassembly of ribosome-RNC complexes. In all, our results highlight the supporting role of the ribosome for newly synthesized protein chains, showing that it establishes interactions with RNCs via specific r-proteins. Results And Discussion Experimental design. This work focuses on ribosome-bound nascent chains (RNCs) derived from Escherichia coli flavohemoglobin (Hmp, Fig. 1 a) and from the phosphorylated insulin receptor interacting region (PIR) of the growth factor receptor-bound protein 14 from Rattus norvegicus (Fig. 1 b). The Hmp protein comprises three domains, an N-terminal heme-binding (domain 1), a flavin adenine dinucleotide-binding (domain 2) and a C-terminal nicotinamide adenine dinucleotide-binding domain (domain 3), as shown in Fig. 1 c 68 . Several RNC chain lengths were examined, and all pertinent constructs are shown as solid bars in Fig. 1 c. Hmp plays a key role in O 2 , NO and CO transport in E. coli , and is involved in a variety of signaling pathways 69 , 70 . The cofactor-free form of Hmp is denoted as apoHmpH. Importantly, previous studies established that the N-terminal globin domain of Hmp is stable and folded even in its apo form 71 . Our second target protein, PIR, is intrinsically disordered 72 , i.e., an IDP (Fig. 1 b). The specific nascent-chain constructs of both proteins analyzed in this work are schematically illustrated in Fig. 1 c,d. Ribosome-nascent-protein interactions were probed with via the well-characterized zero-length chemical crosslinker carbodiimide 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) 51 , 73 , 74 . Nascent chains were chemically crosslinked via known procedures 51 involving a combination of low-pH SDS-PAGE 75 and Western blotting in the absence and presence of the trigger factor (TF) chaperone. Notably, EDC enables the detection of existing noncovalent interactions and it does not provide an accurate quantitation of interacting populations, as discussed at length by Guzman-Luna et al. 51 . Yet, in the presence of appropriate controls, EDC is an extremely valuable tool to detect the existence of protein-protein interactions within the ribosome-nascent-chain complex. In addition, relative changes in the extent of the interactions, for any given ribosome-nascent-chain complex (RNC) as a function of environmental changes (e.g., variable urea or chaperone concentrations) were also qualitatively assessed. In general, due to the established presence of crosslinking-incompetent populations in RNC/r-protein complexes detected via EDC 51 , interacting populations tend to be underestimated. Site-specific fluorescence labeling of nascent proteins at their N terminus enables focusing exclusively on interactions involving the nascent protein. Low-pH-gel and Western-blot were collected to explore interactions between nascent chains and r-proteins. It is worth noting that EDC does not have high accessibility within the exit-tunnel core 51 . Therefore, detection of interactions within the tunnel core is not expected, within our experimental setup. It is also important to mention that, under our experimental conditions, EDC does not report on interactions involving nascent protein chains and ribosomal RNA (rRNA). In the presence of imidazole, crosslinks between RNA 5' phosphate and aliphatic amines of proteins are known to take place 73 . However, our samples did not contain imidazole, and this chemical would anyways be unable to detect interactions not involving the 5’ end of RNA. Therefore, even in the presence of imidazole, EDC would likely underestimate all potential interactions with RNA. Thus, interactions between nascent proteins and rRNA are beyond the scope of this study. The compaction, tumbling rates, size (expressed in terms of approximate number of residues)and local-motion amplitude of non-interacting RNC regions were assessed via fluorescence depolarization in the frequency domain. In this way, it was possible to gain complementary and more comprehensive insights into RNC conformational characteristics. The apparent thermodynamic stability of r-proteins was collectively assessed by Trp fluorescence emission spectroscopy as a function of urea concentration. The apparent stability of the peptidyl transferase center (PTC) of the ribosome in the presence of a variety of RNCs was evaluated by urea titrations upon detection via a puromycin-release assay. Finally, the empty-ribosome and RNC assembly status of the ribosome, in terms of 30S, 50S and 70S subunits, was assessed via sucrose-gradients and negative-staining transmission electron microscopy. ApoHmp RNCs of increasing length interact with ribosomal protein L23. We elected to probe whether apoHmpH RNCs interact with the L23, L24 and L29 r-proteins, which reside within the vestibule of the ribosomal exit tunnel and the adjacent outer surface of the ribosome. We explored the interaction patterns of three representative nascent chains, namely apoHmp 1 − 55 , apoHmp 1 − 140 and apoHmp 1 − 189 . The data for these RNCs are shown in Fig. 2 . A side-by-side comparison between low-pH SDS-Page gels 75 and Western blots indicate that all three nascent proteins interact with ribosomal protein L23. Western blotting carried out with antibodies against ribosomal proteins L24 and L29, shown in the Supplementary Information (Fig. S1 ), indicates no evidence for interactions between the L24 and L29 r-proteins and the target RNCs. Therefore, apoHmpH RNCs interact exclusively with ribosomal protein L23. In contrast, intrinsically disordered PIR 1 − 91 RNCs, analyzed in previous studies 51 , interact with both the L23 and L29 ribosomal proteins. Under our experimental conditions, the fraction of interacting RNCs is different, for nascent chains derived from apoHmp 1 − 55 , apoHmp 1 − 140 , apoHmp 1 − 189 (Fig. 2 a-c) and intrinsically disordered PIR 1 − 91 51 . Indeed, apoHmp 1 − 55 and PIR 1 − 91 crosslink only in part, unlike apoHmp 1 − 140 and apoHmp 1 − 189 RNCs, which are nearly 100% crosslinked. On the other hand, the larger extent of crosslinking of the foldable apoHmp 1 − 140 and apoHmp 1 − 189 RNCs relative to apoHmp 1 − 55 and PIR may be mainly a consequence of the greater number of EDC-reactive residues of apoHmp 1 − 140 (25 EDC-reactive residues, ca. 20 beyond the tunnel core) and apoHmp 1 − 189 (36 EDC-reactive residues, ca. 30 beyond the tunnel core) relative to apoHmp 1 − 55 (11 EDC-reactive residues, ca. 5 beyond the tunnel core) and PIR (14 EDC-reactive residues, ca. 12 beyond the tunnel core). In support of this argument (see sections below), the urea sensitivity of the L23 / RNC complexes is similar for all RNCs, suggesting comparable interaction strengths. Importantly, given that fluorescence anisotropy-decay data (see later sections) show that apoHmp 1 − 140 and apoHmp 1 − 189 RNCs have dynamic and independently tumbling N-terminal compact regions, it is clear that the RNC regions interacting with the ribosomal surface cannot include any significant fraction of N-terminal residues belonging to the compact region. In the case of the longest RNCs analyzed in this work, corresponding to apoHmp 1 − 189 , we found an additional interacting complex of higher molecular weight, which we denote as RP2 (Fig. 2 c). The corresponding population includes r-protein L23, according to Western blotting, see Fig. 2 c, and one additional unidentified protein of c.a. 6–10 kDa, according to molecular weight arguments. Our Western blots indicate that L29 (7 kDa) is not present in the RP2 band (Fig. S1 f). Yet, other cytoplasmic E. coli chaperones and ribosome interactors (GroEL, GroES, SecB, DnaK/DnaJ/GrpE, SRP and ClpB; MW range: 48–80 kDa) are ruled out, as they would appear well above the RP2 complex in our gels (Fig. 2 c and d). Due to its close spatial proximity to L23 (Fig. 3 a,b,c and d) and based on the above-mentioned molecular-weight arguments, it is possible that RP2 comprises both L23 and L29. However, our monoclonal antibodies against r-protein L29 were unable to capture an L29 epitope, as part of the crosslinked complex adsorbed onto the PVDF membrane. To further test for the possibility that L29 being part of the RP2 interacting protein pair, additional experiments in the presence of polyclonal antibodies against L29 will be carried out in the future. The data in Figs. 2 and S2 also show that a fraction of the apoHmp 1 − 55 , apoHmp 1 − 140 and apoHmp 1 − 189 RNCs interacts with the trigger factor (TF) chaperone. The presence of these contacts was assessed upon comparing denaturing gels for data collected with wild-type (WT) and TF-depleted ( Dtig ) E. coli cell strains. Indeed, RNC / TF interactions are already known to exist from previous literature, especially for nascent proteins longer than ca. 100–110 residues 8 42,76−79 Previous studies also showed that TF interacts with client proteins that bear a fairly expanded conformation, in their bound state 80 – 82 . On the other hand, the small observed fraction of apoHmp 1 − 55 interacting with TF (Fig. 2 a) is unexpected. This result implies the presence of a highly stretched conformation of this short (55-residue) RNC, which must reside mostly within the 80–100 Å-long ribosomal exit tunnel. Yet, apoHmp 1 − 55 manages to reach out to the TF chaperone, which is known to dock onto the outer surface of the ribosome via the L23 and L29 r-proteins. This conformational stretching experienced by a small fraction of the short apoHmp 1 − 55 RNC is fascinating and unprecedented. Indeed, the presence of L23-docked and TF-docked apoHmp 1 − 55 nascent-chains suggests that cotranslational conformational sampling can take place even in the case of a fairly short RNC. TF and L23 are known to interact with one another on the ribosome 77 , 83 , 84 , though we presently cannot explicitly discriminate whether the nascent chains interact with L23 and TF, or if the nascent chain interacts with TF, which in turn interacts with L23. Here, we propose the simplest scenario namely that RNCs interact with TF only and, in turn, TF interacts with L23, which is known to be the TF docking site on the ribosome 2 . Finally, in this work we only analyzed the behavior of RNCs in the presence of moderate concentrations of the 70 kDa Hsp70 chaperone. Hsp70 was studied in the context of the DnaK/DnaJ/GrpE chaperone system, denoted here as K/J/E. Now, the wild-type (WT) cell-free system used in Fig. 2 a,b,d contains K/J/E at 0.5, 0.04, 0.05 mM concentrations, respectively, which are significantly lower than physiologically relevant values. Interestingly, at these low K/J/E concentrations, none of these chaperones is bound to the apoHmpH 1 − 55 , apoHmp 1 − 140 and apoHmp 1 − 189 resuspended RNCs, as shown in Fig. 2 a,b,d. Therefore, the Hsp70 chaperone does not bind the RNCs studied in this work. The effect of higher, more physiologically relevant (20–50 mM) K/J/E concentrations will be studied elsewhere 85 . In all, our data show that apoHmp 1 − 55 , apoHmpH 1 − 140 and apoHmp 1 − 189 RNCs interact with either the L23 r-protein alone (apoHmp 1 − 55 and apoHmpH 1 − 140 , Fig. 3 e), with L23 and another ribosomal protein (apoHmpH 1 − 189 ), or with the TF chaperone (all RNCs, including apoHmp 1 − 55 ). We propose that these two classes of interactions (i.e., with r-proteins and with TF) play a similar chaperone-like role. This concept is consistent with previous studies, which showed that the ribosome serves as a nascent-chain solubilizing agent even in the absence of chaperones 7 . The fairly solvent-exposed nonpolar patch of the L23 r-protein, highlighted in Fig. 3 c-d, is also consistent L23 being able to interact with nonpolar regions of RNCs. Future work will focus on genomic E. coli r-protein modifications aimed at disrupting the detected interactions. Ribosome-bound apoHmp nascent chains of variable length have a compact N-terminal region. Next, we performed fluorescence depolarization decay experiments in the frequency domain 86 – 88 to probe the rotational dynamics of nascent chains encoding foldable sequences. This technique has been previously employed to assess the rotational correlation time (t c ) and amplitude of rotational motions of RNCs 7 , 20 , 21 , 52 , 89 . The goal of this experiment was to determine whether RNCs harboring long nascent chains display any degree of compaction. We focused on RNCs of apoHmp 1 − 140 , corresponding to the N-terminal domain 1 of Hmp (Fig. 1 a), and RNCs of apoHmp 1 − 189 , which comprise Hmp’s domain 1 and an additional 49 C-terminal residues belonging to domain 2 (Fig. 1 c). Nascent proteins were site-specifically labeled at their N terminus with the BODIPY-FL fluorophore as described 20 . Once information on nascent-chain compaction is in hand, the interplay between ribosome and nascent-chain interactions, and their sensitivity to urea denaturation can be more rationally explored and understood, as apparent in the sections below. Representative data for apoHmp 1 − 140 and apoHmp 1 − 189 are shown in panels a and b of Fig. 4 , respectively. Both RNCs display informative frequency-domain anisotropy decay profiles. As shown in Fig. 4 c and consistent with the very low reduced c 2 values, the fits that include 3 rotational-tumbling components give the best results. Importantly, panels c and d of Fig. 4 show that both apoHmp 1 − 140 and apoHmp 1 − 189 RNCs are characterized by an N-terminal compact domain that tumbles independently from the ribosome. This conclusion was reached upon applying known procedures based on a combination of microscale viscosity and fluorescence depolarization in the frequency domain. In both cases, this domain spans ca. 63 to 94 residues, depending on the exact shape. Note that RNC shape assessment is beyond the scope of this work. Regardless of the actual overall morphology of the compact domains, the fact that a compact domain of identical size is observed for both apoHmp 1 − 140 and apoHmp 1 − 189 suggests that both constructs undergo a similar degree of partial folding on the ribosome. Surprisingly, the observed size of the compact domain of apoHmp 1 − 189 RNCs is significantly smaller than the size of the entire apoHmp domain 1, which comprises 140 residues (Fig. 1 c). Therefore, biosynthesis of the additional 49 C-terminal amino acids belonging to domain 2 is not sufficient to lead to complete folding of the N-terminal domain domain 1, for this protein. In addition, cone semi-angle analysis of the fluorescence anisotropy decay data (Fig. 4 c) shows that the compact domain of Hmp 1 − 189 RNCs spans a slightly wider cone semi-angle (26.5⁰ ± 0.5⁰) than Hmp 1 − 140 RNCs (20⁰ ± 0.2⁰), consistent with the fact that the latter construct likely projects slightly further out from the ribosomal surface than the shorter Hmp 1 − 140 construct. In all, our fluorescence anisotropy data show that the Hmp 1 − 140 and Hmp 1 − 189 nascent chains are both comparably compact and no more than partially folded, while on the ribosome, with Hmp 1 − 189 spanning a slightly wider cone semi-angle. All the above information on fluorescence anisotropy decays is pictorially recapitulated by the cartoons of Fig. 4 e. The images presented in this figure also show a variety of compact species that take into account the r-protein-interacting and non-interacting populations deduced from the SDS-Page gels and Western blotting data of Fig. 2 . In order to recapitulate the RNC/r-protein interaction profiles and nascent-protein conformation knowledge gained so far, a model highlighting the leading trends is shown in Fig. 5 . The RNCs displayed in this figure highlight the evolution of foldable apoHmpH nascent-chain interactions with r-proteins as a function of chain elongation. Briefly, when the nascent chain reaches a 55-residue length, the main detected interactions are with r-protein L23. No compact region is shown at this chain length, consistent with known fluorescence anisotropy-decay data collected on RNCs of a related globin 20 . As the nascent chain gets longer and reaches a length of 140 residues, interactions with L23 are still present, but the chain also features a non-interacting compact region that spans a cone semi-angle of ca. 20⁰. As the nascent chain reaches 189-residue length, two classes of RNC populations interacting with r-proteins are present. The former interacts only with L23 and the other one also interacts with an additional ribosomal protein. In both cases, an N-terminal compact region encompassing 65–94 residues is also detected. Further chain elongation and ribosome-release processes, which are beyond the scope of this study, are expected to give rise to the full-length ribosome-released folded protein. Nascent chain-L23 complexes have the same apparent stability regardless of RNC sequence. To further explore the nature of the interactions between the L23 r-protein and nascent chains of increasing length and variable sequence, we performed urea titrations with chemical crosslinking detection (Fig. 6 a,b). EDC readily reacts with amines and carboxylic acid functional groups, and there is no loss of EDC reactivity even in the presence of high urea concentrations 90 . It is worth noting that the interactions identified in this work are not induced by the covalently N-terminal-linked BODIPY-502 fluorophore, as previous work has shown that this fluorophore does not interact with resuspended ribosomes under conditions like those of the present study 20 . Therefore, by unfolding the complex in the presence of urea and subsequently adding EDC, we expect to gain insights into the urea sensitivity of nascent chain-L23 complexes. While different RNC constructs are expected to bear a different number of EDC-reactive residues, denaturant titration of RNC complexes always examine the same nascent chain at variable urea concentration. Therefore, it is not necessary to normalize the data on a per-EDC-reactive-residue basis, as done in other studies 51 . After collecting gel data on representative apoHmp and PIR nascent chains (Fig. 6 c), we estimated the apparent stability (ΔG° app,unfold ) of nascent chain-L23 complexes following a known extrapolation method which is further described in the SI methods 91 . Representative EDC-mediated urea titrations are shown in Fig. 6 d. Corresponding plots and apparent-stability data are displayed in Fig. 6 e,f. The matching two-tailed Student’s t-test is provided in Fig. 6 g. As shown in Fig. 6 h, the apparent stability values for the apoHmp and PIR nascent-chain/L23 complexes (RP1 complexes) range between DG 0 app,unf of 2.8 ± 1.3 and 5.8 ± 1.2 kcal mol − 1 . As shown in the t-test of Fig. 6 g, all complexes display the same apparent stability within error. The corresponding values for the apparent unfolding equilibrium constants K app (Table S2) are within the 590 ± 340 mM to 58 ± 41 mM range. These values, if regarded as estimates of the lower limits of the expected dissociation constants of r-protein/RNC complexes, suggest that the binding affinity of the apoHmp and PIR nascent-chain/L23 complexes (RP1 complexes) is overall rather weak. This qualitative estimate is consistent with the need for the interactions to be continuously remodeled during translation elongation. Interestingly, the observed trends apply even though the nascent-chain portions emerging from the ribosomal exit-tunnel core have widely different nonpolar and net-charge-per-residue (Fig. 6 i) as well as widely different total nonpolar surface accessible surface-area values (Fig. 6 j). In summary, the urea-titrations in Fig. 6 show that the urea sensitivity of r-protein-nascent-chain complexes is similar regardless of the nature and length of the nascent chain, across the short and long (55- to 189-residue) chains examined here. In other words, RNC/-r-protein complexes have the same apparent stability, even though the RNCs have widely different physical properties and compaction (as discussed above and) and in the case of PIR, lack of compaction (discussed in previous work). 51 , 52 Given that the amino-acid sequences of the interacting regions of apoHmp 1 − 55 and apoHmp 1 − 140 must be different yet the interactions are of comparable apparent strength, the contacts are likely to be of nonspecific nature (Fig. 6 f-h). This scenario, again, is consistent with the fact RNC-r-protein interactions likely need continuous remodeling during translation elongation. Finally, the urea titrations described in Fig. 6 are highly informative, as they also display the urea dependence of complexes between nascent chains and the trigger factor chaperone (RNC/TF complexes), e.g., see selected upper bands in Fig. 6 d. The quality of the data for the RNC/TF complexes was rather poor due to unreliable pre-transition baselines, therefore we did not deduce apparent stability values. On the other hand, as shown in Fig. 6 d and in the plots of Fig. S3, the complexes with the TF chaperones are consistently less stable than the corresponding complexes with the L23 protein. This result suggests that the interactions between RNCs and the TF chaperone are even weaker than the interactions between RNCs and the L23 r-protein. Hence, nascent chains interacting with TF may in general be allowed more extensive conformational sampling in their bound state than nascent chains interacting with r-proteins. Additional future work will be devoted to further explore this hypothesis. Nascent chain and r-protein interaction strength does not vary in the presence of one or more molecular chaperone. Next, we explored the effect of molecular chaperones TF and Hsp70 on the RNC-r-protein interactions via the same type of EDC-mediated urea titrations employed in the last section. The effect of Hsp70 was examined in the context of the K/J/E chaperone system. TF is known to associate with prokaryotic ribosomes 92 and K/J/E works in cooperation with TF 93 to promote nascent-protein folding and prevent nascent-protein aggregation 8 , 84 , 94 , 95 . First, we evaluated apoHmp 1 − 189 devoid of both TF and the Hsp70 chaperone system (K/J/E), apoHmp 1 − 189 in the presence of low concentrations of TF (2–15 nM) only, apoHmp 1 − 189 in the presence of low concentrations of K/J/E (0.5, 0.04 and 0.05 mM, respectively) only, and apoHmp 1 − 189 in the presence of both chaperones at low concentration (Fig. 7 a). Urea titrations were carried out with increasing concentrations of urea (Fig. 7 b), and the intensities of the crosslinked fractions were plotted (Fig. 7 c). We then obtained a ΔG° app, unfold values for each of these constructs (Fig. 7 d) and evaluated them with a two-tailed Student’s t-test (Fig. 7 g), similarly to what done for the data in Fig. 6 . Interestingly, the apparent strength of the L23-nascent chain complex was found to be statistically similar in all cases, regardless of chaperone concentration (Fig. 7 f, h). Given that this effect is not due to a variation in the fraction of crosslinked nascent chains to r-proteins (Fig. 7 e, f), via Western Blot analysis, we conclude that the extent of interactions between nascent chains and L23 remains similar in the absence and presence of the TF and K/J/E chaperones (Fig. 2 c). This finding suggests that nascent chains interact with ribosomal L23 in a structurally similar fashion regardless of the absence or presence of chaperones. RNC/r-protein interactions are attenuated at high chaperone levels in a chain-length-dependent manner. To further elucidate the nature of RNC/chaperone complexes, we performed experiments at low (2–15 nM) and high (8 µM) TF concentrations (Fig. 8 ). The high concentration values are representative of physiologically relevant TF concentrations, upon taking into account the differences in the concentrations of actively translating ribosomes in our cell-free system and in live E. coli cells 51 . Interestingly, interactions with r-protein L23 are mostly displaced by interactions with TF, at high TF concentrations (Fig. 8 ). This effect, however, is more pronounced for longer RNCs, as shown by the representative gels of Fig. 8 a,c,e, Fig. S2, and by the comprehensive analysis of the interacting populations shown in Fig. 8 b,d,f. The shortest nascent chains of apoHmp 1 − 55 only show c.a. 25% interactions with TF, even at high TF concentrations. We attribute this result to the fact that apoHmp 1 − 55 is likely too short to form extensive interactions with the TF chaperone. To summarize, in the absence of TF (Figs. 7 and S2), the nascent chain either interacts primarily with r-proteins, mainly, L23. At higher, physiologically relevant concentrations of TF (8 µM TF), RNC interactions with r-proteins are displaced by interactions with this molecular chaperone. It is worth noting that TF is shared with thousands of additional cellular proteins in vivo , unlike in the experiments shown here, which include purified resuspended RNCs. Further, our RNC concentrations are only 20–30 nM. Hence, the TF chaperone is in large excess over RNCs even at the low chaperone concentrations employed here. This scenario differs from the cellular environment where both RNCs and molecular chaperones are at comparable concentrations, within the low uM range. Therefore, we propose that the actual cellular milieu likely involves RNC populations that interact in part with TF and in part with r-proteins. In all, our findings highlight the prominent role of the ribosome as an RNC interactor and suggest that the ribosome may have played a primordial chaperone role in Nature, before the evolution of the TF molecular chaperone. The presence of very short nascent chains stabilizes the 70S ribosomal complex. After exploring nascent chain and r-protein interactions, we investigated the potential effect of these contacts on the bacterial ribosome. We began by performing a series of qualitative sucrose-gradient studies on E. coli empty ribosomes and nascent-chain-loaded ribosomes. Our results, detailed in Fig. S4 and S5, showed that empty-70S ribosomes are more sensitive to urea denaturation than ribosomes bearing tRNAs linked to longer nascent chains. These results agree with previous sucrose gradient studies on RNCs 37 . It appears that the snc-tRNA is responsible for most of the stabilizing effect (Figs. S4 and S5). Interestingly, these data suggest that length and amino-acid sequence of the nascent protein does not influence the urea sensitivity of ribosome-RNC complexes. The peptidyl transferase center site is largely unaffected by nascent-chain sequence and length, beyond 32 residues. Next, we probed whether nascent chains of different length, amino-acid sequence and foldability affect the apparent stability of specific regions of the ribosome. We directed our focus on the peptidyl transferase center (PTC) of the E. coli ribosome, and we explored its urea sensitivity via a nascent-chain ribosome-release assay mediated by puromycin. These experiments employed a larger set of RNCs than in the previous sections. The results of puromycin-release-detected urea titrations are shown in Fig. S8 and further described in the Supplementary Information. Overall, the data show that the apparent stability of the ribosomal PTC is not affected by the presence of nascent chains longer than 32 residues. The global urea sensitivity of ribosomal proteins is largely unaffected by nascent-chain sequence and length. Next, we explored the effect of nascent-chain properties on the overall apparent stability of r-proteins via urea titrations based on Trp fluorescence emission. Trp is a well-known fluorescent reporter, and its emission properties are highly environmentally sensitive. Urea titrations were carried out and Trp fluorescence emission was monitored (Fig. S9b,c). Spectral shifts were regarded as reporters of r-protein folding, and centers of mass of emission spectra were assessed to generate titration curves reporting on the urea sensitivity of r-proteins. Note that incubation time totaling the measurements from beginning and end of experiments did not change the spectral center of mass (Fig. S9d). Urea titration data were processed according to Santoro and Bolen 91 , 96 . Individual representative titration curves are shown in Fig. S9e. The ΔG° app, unfold for each construct are plotted in Fig. S9f and corresponding t-test values are tabulated in Fig. S9g. Nearly all the constructs show statistically similar results, with ΔG° app, unfold values ranging from 2 to 5 kcal•mol − 1 Hence, the presence of peptidyl tRNA, regardless of nascent-chain characteristics, does not affect the urea sensitivity of r-proteins. As shown in previous sections, some nascent chains interact with the specific ribosomal protein L23. On the other hand, these interactions are not sufficiently strong to be detected via this assay, which monitors the overall sensitivity to urea of all r-proteins. Conclusions. The presence of interactions between nascent chains bearing a foldable amino-acid sequence (with no signal or arrest tags) and specific ribosomal proteins has been suggested but never experimentally demonstrated, to date. Here, we identify the ribosomal protein L23 as a specific nascent-chain-interacting partner. L23 establishes noncovalent contacts with nascent chains of the multi-domain foldable model protein apoHmp, which lacks signal/arrest sequences. As nascent chains elongate, the RNC interaction network expands to another ribosomal protein. A non-interacting N-terminal compact RNC region comprising 63–95 residues has also been identified for nascent chains bearing both 140 and 189 residues. A model recapitulating the presence of both RNC/r-protein interactions and non-interacting N-terminal regions is shown in Fig. 5 . Interactions with the TF take over, at high TF chaperone concentrations. Interestingly, ribosomal-protein/nascent-chain complexes have a similar weak apparent stability regardless of nascent-chain sequence, length and degree of foldability. Therefore, we propose that r-proteins shield nascent foldable proteins from aggregation before intramolecular folding becomes thermodynamically favorable, during and(or) immediately after translation. These findings are significant because they unveil the presence of interactions between a foldable nascent chain and the L23 ribosomal protein. In addition, the data reveal that these interactions coexist with nascent-chain compaction across the N-terminal region, suggesting ribosome-facilitated aggregation-prevention and conformational sampling. Materials And Methods Preparation of empty ribosomes. Empty ribosomes were generated from an in-house - prepared A19 WT or A19 Δtig E. coli S30 cell extract as described 20 , 97 . Briefly, cells were grown in Luria-Bertani (LB) broth and harvested at mid-log phase (A 600 ~ 0.6). The cells were lysed through a French press (thermo Electron Corporation, Waltham, MA) at ~ 12,000 psi with a single passage. The lysate was subject to centrifugation at 30910 g and 20°C for 20 min. After centrifugation, the supernatant was incubated in translation buffer (0.75 M Tris-HCl pH 8.2, 7.5 mM DTT, 21 mM Mg(OAc) 2 , 500 µM amino acids, 6 mM ATP, 67 mM PEP and 160 µg •mL − 1 pyruvate kinase) for 80 min to remove any endogenous mRNA from ribosomes. The supernatant was then dialyzed (12–14 kDa MWCO) in buffer (10 mM Tris-HCl pH 8.2, 14 mM Mg(OAc) 2 , 60 mM KOAc and 1 mM DTT) for 12 hrs, with a buffer exchange every 4 hours. The resulting A19 cell extract was used as the empty-ribosome sample. Preparation of RNCs. RNCs were generated using an in-house prepared A19 E. coli transcription-translation coupled cell-free system 20 , 97 as described. Cell strains either including (WT) or lacking ( Δtig ) the trigger factor gene were employed 20 , 97 . Hsp70 chaperone activity was suppressed via the KLR-70 peptide 98 to a final concentration of 0.2 mM. Transcription-translation proceeded for 30 min at 37°C in the presence of BODIPY-FL-Met-tRNA f−Met to specifically label RNCs at the N terminus. BODIPY-FL-Met-tRNA f−Met was prepared as described 20 . RNCs were stalled at various lengths to generate the desired apoHmp and PIR constructs via oligodeoxynucleotide-directed mRNA cleavage 20 , 99 , 100 . An anti-ssrA oligonucleotide 20 was added to a final concentration of 12.83 pmol µL − 1 to prevent premature release of stalled RNCs. RNC pellets were isolated via a sucrose cushion (1.1 M sucrose, 20 mM tris base, 10 mM Mg(OAc) 2 , 500 mM NH 4 Cl, and 0.5 mM EDTA, 1 mM DTT, pH 7.0, as described) 20 and subjected to ultracentrifugation at 160,000 g for 1 hr at 4°C. The purified pellet was dissolved in resuspension buffer (10 mM tris-HCl, 10 mM Mg(OAc) 2 , 60 mM NH 4 Cl, 0,5 mM EDTA and 1.0 mM DTT, pH 7.0) by shaking in an orbital shaker at 200 rpm on ice for 1 hr. Statistics and reproducibility . Statistical data analysis was performed with Excel V. 16.70 software. Data are displayed as the mean with ± the standard error (SE), with the number of independent experiments listed in parenthesis, (e.g., n = 2). Statistically meaningful differences between sets of data were determined via the two-tailed Student t-test. Pairs of results were regarded as statistically different if bearing P values < 0.05. Other experimental procedures. Experimental details on frequency-domain fluorescence-anisotropy, sucrose gradients, low-pH gels, puromycin assays, chemical crosslinking and urea titrations are available in the Supplementary Information. Declarations Data Availability The data that support the findings of the study are available from the corresponding author, S.C., upon reasonable request. Acknowledgments We are thankful to M. Dalphin for helpful discussions. This work was funded by the National Science Foundation (NSF) grants MCB-1616459 and MCB-0951209 (to S.C). M. M. M. and R.B.H. received NIH TEAM-Science Fellowships from the University of Wisconsin-Madison and M.M.M received the Straka Fellowship from the University of Wisconsin-Madison. A. E. V. received a National Science Foundation GRFP graduate fellowship and a Science and Medicine Graduate Research Scholars Fellowship from the University of Wisconsin-Madison. Competing Interests The authors declare no competing interests. Author Contributions M.M. designed and performed experiments, analyzed the data including statistical analysis, and wrote the manuscript. V.G.L. performed Western blotting experiments and matching data analysis. A.V. contributed to method development and participated in the writing of early versions of the manuscript. R.H. performed fluorescence depolarization experiments and took care of matching data analysis. A.S. participated in method development. W.W. contributed to figure preparation and design and helped with urea titration experiments. S.C. designed the project, participated in data analysis, wrote the manuscript and contributed to manuscript editing. A.F. performed some of the Western blotting experiments. References Wilson, D. N. & Beckmann, R. The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling. Curr. Opin. Struct. Biol. 21, 274–282, (2011). Kramer, G., Boehringer, D., Ban, N. & Bukau, B. The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat. Struct. Mol. Biol. 16, 589, (2009). Fedyukina, D. V. & Cavagnero, S. Protein Folding at the Exit Tunnel. Annu. Rev. Biophys. 40, 337–359, (2011). Pechmann, S., Willmund, F. & Frydman, J. The ribosome as a hub for protein quality control. Mol. Cell 49, 411–421, (2013). Liutkute, M., Samatova, E. & Rodnina, M. V. Cotranslational folding of proteins on the ribosome. Biomolecules 10, 97, (2020). Waudby, C. A., Dobson, C. M. & Christodoulou, J. Nature and Regulation of Protein Folding on the Ribosome. Trends Biochem. Sci 44, 914–926, (2019). Addabbo, R. M. et al. Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis. J. Phys. Chem. B 124, 6488–6507, (2020). Mecha, M. F., Hutchinson, R. B., Lee, J. H. & Cavagnero, S. Protein folding in vitro and in the cell: From a solitary journey to a team effort. Biophys. Chem. 287, 106821, (2022). Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920, (2000). Harms, J. et al. High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107, 679–688, (2001). Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis. Science 289, 920–930, (2000). Voss, N., Gerstein, M., Steitz, T. & Moore, P. The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893–906, (2006). Malkin, L. I. & Rich, A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J. Mol. Biol. 26, 329–346, (1967). Blobel, G. & Sabatini, D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions: I. Location of the polypeptides within ribosomes. J. Cell Biol. 45, 130–145, (1970). Wang, S., Sakai, H. & Wiedmann, M. NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. J. Cell Biol. 130, 519–528, (1995). Kramer, G., Ramachandiran, V. & Hardesty, B. Cotranslational folding—omnia mea mecum porto? The international journal of biochemistry & cell biology 33, 541–553, (2001). Tsalkova, T., Odom, O., Kramer, G. & Hardesty, B. Different conformations of nascent peptides on ribosomes. J. Mol. Biol. 278, 713–723, (1998). Woolhead, C. A., McCormick, P. J. & Johnson, A. E. Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins. Cell 116, 725–736, (2004). Fedyukina, D. V., Jennaro, T. S. & Cavagnero, S. Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms. J. Biol. Chem. 289, 6740–6750, (2014). Ellis, J. P., Bakke, C. K., Kirchdoerfer, R. N., Jungbauer, L. M. & Cavagnero, S. Chain dynamics of nascent polypeptides emerging from the ribosome. ACS Chem. Biol. 3, 555–566, (2008). Hutchinson, R. B., Chen, X., Zhou, N. & Cavagnero, S. Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins. J. Phys. Chem. B 125, 6543–6558, (2021). Ellis, J. P., Culviner, P. H. & Cavagnero, S. Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions. Protein Sci. 18, 2003–2015, (2009). Lu, J. & Deutsch, C. Secondary Structure Formation of a Transmembrane Segment in Kv Channels. Biochemistry 44, 8230–8243, (2005). Mingarro, I., Nilsson, I., Whitley, P. & Von Heijne, G. Different conformations of nascent polypeptides during translocation across the ER membrane. BMC Cell Biol. 1, 3, (2000). Bhushan, S. et al. α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 17, 313, (2010). Woolhead, C. A., McCormick, P. J. & Johnson, A. E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the dribosome and in their exposure to ribosomal proteins. Cell 116, 725–736, (2004). Agirrezabala, X. et al. A switch from α-helical to β-strand conformation during co-translational protein folding. The EMBO Journal 41, e109175, (2022). Kosolapov, A. & Deutsch, C. Tertiary interactions within the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 16, 405–411, (2009). Nilsson, O. B. et al. Cotranslational folding of spectrin domains via partially structured states. Nat. Struct. Mol. Biol. 24, 221–225, (2017). Tian, P. et al. Folding pathway of an Ig domain is conserved on and off the ribosome. Proc. Natl. Acad. Sci. U. S. A. 115, E11284, (2018). Holtkamp, W. et al. Cotranslational protein folding on the ribosome monitored in real time. Science 350, 1104–1107, (2015). Liutkute, M., Maiti, M., Samatova, E., Enderlein, J. & Rodnina, M. V. Gradual compaction of the nascent peptide during cotranslational folding on the ribosome. Elife 9, (2020). Ziv, G., Haran, G. & Thirumalai, D. Ribosome exit tunnel can entropically stabilize α-helices. Proc. Natl. Acad. Sci. U. S. A. 102, 18956, (2005). Marino, J., von Heijne, G. & Beckmann, R. Small protein domains fold inside the ribosome exit tunnel. FEBS Lett. 590, 655–660, (2016). Bañó-Polo, M. et al. Transmembrane but not soluble helices fold inside the ribosome tunnel. Nat. Commun. 9, 5246, (2018). Wruck, F. et al. The ribosome modulates folding inside the ribosomal exit tunnel. Commun. Biol. 4, (2021). Samelson, A. J., Jensen, M. K., Soto, R. A., Cate, J. H. D. & Marqusee, S. Quantitative determination of ribosome nascent chain stability. Proc. Natl. Acad. Sci. U. S. A. 113, 13402–13407, (2016). Bhushan, S. et al. Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide. Mol. Cell 40, 138–146, (2010). Eisner, G., Moser, M., Schäfer, U., Beck, K. & Müller, M. Alternate Recruitment of Signal Recognition Particle and Trigger Factor to the Signal Sequence of a Growing Nascent Polypeptide. J. Biol. Chem. 281, 7172–7179, (2006). Houben, E. N. G., Zarivach, R., Oudega, B. & Luirink, J. Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome . Vol. 170 (2005). Peterson, J. H., Woolhead, C. A. & Bernstein, H. D. The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding. Mol. Microbiol. 78, 203–217, (2010). Ullers, R. S. et al. Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome. J. Cell Biol. 161, 679–684, (2003). Bhushan, S. et al. SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center. PLoS Biol. 9, 10, (2011). Cruz-Vera, L. R., Rajagopal, S., Squires, C. & Yanofsky, C. Features of Ribosome-Peptidyl-tRNA Interactions Essential for Tryptophan Induction of tna Operon Expression. Mol. Cell 19, 333–343, (2005). Nakatogawa, H. & Ito, K. The ribosomal exit tunnel functions as a discriminating gate. Cell 108, 629–636, (2002). Seidelt, B. et al. Structural Insight into Nascent Polypeptide Chain–Mediated Translational Stalling. Science 326, 1412–1415, (2009). Carragher, B. et al. Current outcomes when optimizing ‘standard’sample preparation for single-particle cryo‐EM. J. Microsc. 276, 39–45, (2019). Zhang, Y., Wolfle, T. & Rospert, S. Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae. J. Biol. Chem. 288, 33697–33707, (2013). Burridge, C. et al. Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy. Chem. Sci. 12, 13120–13126, (2021). Waudby, C. A., Burridge, C. & Christodoulou, J. Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex. J. Magn. Reson. 326, (2021). Guzman-Luna, V., Fuchs, A. M., Allen, A. J., Staikos, A. & Cavagnero, S. An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel. Commun. Biol. 4, 1–17, (2021). Knight, A. M. et al. Electrostatic Effect of the Ribosomal Surface on Nascent Polypeptide Dynamics. ACS Chem. Biol. 8, 1195–1204, (2013). Cabrita, L. D. et al. A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Nat. Struct. Mol. Biol. 23, 278–285, (2016). Cabrita, L. D., Hsu, S. T. D., Launay, H., Dobson, C. M. & Christodoulou, J. Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 106, 22239–22244, (2009). Hsu, S. T. D., Cabrita, L. D., Fucini, P., Christodoulou, J. & Dobson, C. M. Probing Side-Chain Dynamics of a Ribosome-Bound Nascent Chain Using Methyl NMR Spectroscopy. J. Am. Chem. Soc. 131, 8366-+, (2009). Subramanian, A. R., Davis, B. D. & Beller, R. J. in Cold Spring Harbor symposia on quantitative biology . 223–230 (Cold Spring Harbor Laboratory Press). Beller, R. J. & Davis, B. D. Selective dissociation of free ribosomes of Escherichia coli by sodium ions. J. Mol. Biol. 55, 477–485, (1971). Edelman, I. S., Ts'o, P. O. & Vinograd, J. The binding of magnesium to microsomal nucleoprotein and ribonucleic acid. Biochim. Biophys. Acta 43, 393–403, (1960). Van Der Saag, P. T., Vlak, J. M. & De Greef, T. F. Ribosomes from Xenopus laevis eggs and embryos in a cell-free protein-synthesizing system: translational regulation. Cell Differ. 4, 385–397, (1976). Ron, E. Z., Kohler, R. E. & Davis, B. D. Magnesium ion dependence of free and polysomal ribosomes from Escherichia coli. J. Mol. Biol. 36, 83–89, (1968). Oppenheim, J., Scheinbuks, J., Biava, C. & Marcus, L. Polyribosomes in Azotobacter vinelandii: I. Isolation, characterization and distribution of ribosomes, polyribosomes and subunits in logarithmically growing Azotobacter. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 161, 386–401, (1968). Kelley, W. S. & Schaechter, M. Magnesium ion-dependent dissociation of polysomes and free 70 s ribosomes in Bacillus megaterium. J. Mol. Biol. 42, 599–602, (1969). Beller, R. J. & Lubsen, N. H. Effect of polypeptide chain length on dissociation of ribosomal complexes. Biochemistry 11, 3271–3276, (1972). Spirin, A. S. Structural transformations of ribosomes (dissociation, unfolding and disassembly). FEBS Lett. 40, S28-S37, (1974). Spitnik-Elson, P., Greeman, B. & Abramovitz, R. The Influence of 6-M Urea on 30-S Ribosomes of Escherichia coli. Eur. J. Biochem. 49, 87–92, (1974). Roberts, M. E. & Walker, I. O. Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 199, 184–193, (1970). Roberts, M. E. & Walker, I. Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis 199, 184–193, (1970). Ilari, A., Bonamore, A., Farina, A., Johnson, K. A. & Boffi, A. The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket. J. Biol. Chem. 277, 23725–23732, (2002). Bonamore, A. & Boffi, A. Flavohemoglobin: Structure and reactivity. Iubmb Life 60, 19–28, (2008). Forrester, M. T. & Foster, M. W. Protection from nitrosative stress: A central role for microbial flavohemoglobin. Free Radic. Biol. Med. 52, 1620–1633, (2012). Eun, Y. J., Kurt, N., Sekhar, A. & Cavagnero, S. Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin. J. Mol. Biol. 376, 879–897, (2008). Moncoq, K. et al. The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling. FEBS Lett. 554, 240–246, (2003). Hermanson, G. (Elsevier, 2013). Hoare, D. t. & Koshland, D. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J. Biol. Chem. 242, 2447–2453, (1967). Kirchdoerfer, R. N., Huang, J. J. T., Isola, M. K. & Cavagnero, S. Fluorescence-based analysis of aminoacyl- and peptidyl-tRNA by low-pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 364, 92–94, (2007). Raine, A., Lovmar, M., Wikberg, J. & Ehrenberg, M. n. Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences. J. Biol. Chem. 281, 28033–28038, (2006). Ferbitz, L. et al. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590–596, (2004). Lakshmipathy, S. K. et al. Identification of nascent chain interaction sites on trigger factor. J. Biol. Chem. 282, 12186–12193, (2007). Oh, E. et al. Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo. Cell 147, 1295–1308, (2011). Saio, T., Guan, X., Rossi, P., Economou, A. & Kalodimos, C. G. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344, 1250494, (2014). Nilsson, O. B., Müller-Lucks, A., Kramer, G., Bukau, B. & von Heijne, G. Trigger factor reduces the force exerted on the nascent chain by a cotranslationally folding protein. Journal of molecular biology 428, 1356–1364, (2016). Deckert, A. et al. Structural characterization of the interaction of alpha-synuclein nascent chains with the ribosomal surface and trigger factor. Proc. Natl. Acad. Sci. U.S.A 113, 5012–5017, (2016). et al. Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor. Proc. Natl. Acad. Sci. U.S.A 113, 5012–5017, (2016). Deuerling, E., Schulze-Specking, A., Tomoyasu, T., Mogk, A. & Bukau, B. Trigger factor and DnaK cooperate in folding of newly synthesized proteins. Nature 400, 693–696, (1999). Hesterkamp, T. & Bukau, B. Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli. The EMBO Journal 17, 4818–4828, (1998). Beechem, J. M. & Gratton, E. in Time-Resolved Laser Spectroscopy in Biochemistry (ed J.R. Lakowicz) 70–81 (1988). Jameson, D. M., Gratton, E. & Hall, R. D. The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry. Appl. Spectrosc. Rev. 20, 55–106, (1984). Ross, J. A. & Jameson, D. M. Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence. Photochem. Photobiol. Sci. 7, 1301–1312, (2008). Weinreis, S. A., Ellis, J. P. & Cavagnero, S. Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome. Methods 52, 57–73, (2010). Lewis, S. D. & Shafer, J. A. Conversion of exposed aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues. Biochimica et Biophysica Acta (BBA) - Protein Structure 303, 284–291, (1973). Santoro, M. M. & Bolen, D. W. Unfolding free-energy changes determined by the linear extrapolation method.1.unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 8063–8068, (1988). Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332, (2011). Agashe, V. R. et al. Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199–209, (2004). Teter, S. A. et al. Polypeptide Flux through Bacterial Hsp70: DnaK Cooperates with Trigger Factor in Chaperoning Nascent Chains. Cell 97, 755–765, (1999). Wruck, F. et al. Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches. J. Mol. Biol. 430, 438–449, (2018). Pace, C. N. Measuring and increasing protein stability. Trends Biotechnol. 8, 93–98, (1990). Bakke, C. K., Jungbauer, L. M. & Cavagnero, S. In vitro expression and characterization of native apomyoglobin under low molecular crowding conditions. Protein Expr. Purif. 45, 381–392, (2006). Dalphin, M. D., Stangl, A. J., Liu, Y. & Cavagnero, S. KLR-70: A Novel Cationic Inhibitor of the Bacterial Hsp70 Chaperone. Biochemistry 59, 1946–1960, (2020). Behrmann, M. et al. Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli. J. Biol. Chem. 273, 13898–13904, (1998). Donis-Keller, H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 7, 179–192, (1979). Yaeger-Weiss, S. K. et al. Net charge and nonpolar content guide the identification of folded and prion proteins. Biochemistry 59, 1881–1895, (2020). Tsodikov, O. V., Record, M. T., Jr. & Sergeev, Y. V. Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem. 23, 600–609, (2002). Additional Declarations No competing interests reported. Supplementary Files MerribonetworksupSCIREP040923.docx Cite Share Download PDF Status: Published Journal Publication published 29 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 06 Jul, 2023 Reviews received at journal 03 Jul, 2023 Reviewers agreed at journal 03 Jul, 2023 Reviews received at journal 04 May, 2023 Reviewers agreed at journal 23 Apr, 2023 Reviewers agreed at journal 18 Apr, 2023 Reviewers invited by journal 18 Apr, 2023 Editor assigned by journal 12 Apr, 2023 Editor invited by journal 12 Apr, 2023 Submission checks completed at journal 12 Apr, 2023 First submitted to journal 24 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2734168","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":190959527,"identity":"b5dcf4aa-6957-40cc-be1a-7655ca32a045","order_by":0,"name":"Meranda M. Masse","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meranda","middleName":"M.","lastName":"Masse","suffix":""},{"id":190959528,"identity":"eafd6943-eb9f-4b07-b07d-74e030459fe9","order_by":1,"name":"Valeria Guzman-Luna","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Guzman-Luna","suffix":""},{"id":190959529,"identity":"8631d1ea-6be0-4312-8b5b-80db150a91ee","order_by":2,"name":"Angela E. Varela","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Angela","middleName":"E.","lastName":"Varela","suffix":""},{"id":190959530,"identity":"e9267875-a72b-4355-8058-dba0953877c1","order_by":3,"name":"Rachel B. Hutchinson","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"B.","lastName":"Hutchinson","suffix":""},{"id":190959531,"identity":"1847bb8f-586c-457e-976e-2a4a24bf8e7b","order_by":4,"name":"Aniruddha Srivastava","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aniruddha","middleName":"","lastName":"Srivastava","suffix":""},{"id":190959532,"identity":"437f0b9c-a5ba-46e7-8fff-4f5472fdb5b0","order_by":5,"name":"Wanting Wei","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wanting","middleName":"","lastName":"Wei","suffix":""},{"id":190959533,"identity":"7f142536-5ac3-4fca-85ef-795e89333700","order_by":6,"name":"Andrew M. Fuchs","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"M.","lastName":"Fuchs","suffix":""},{"id":190959534,"identity":"2bb0caf2-940e-44e2-b09b-89148cbc05c5","order_by":7,"name":"Silvia Cavagnero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYFAD9gY2MI1MEgA8B0jWIpGArAyPFv72M4Yfv+6wyeeXfPzswccchsQ+/sMPGD6UHcZt9pkcY2nZM2mWM2enmRvO3MaQ2CaRZsA44xxuLQYMaQnSkm2HDQxu57BJ825jyG2T4GFg5m3Do4X/WfJvybb/BgY3z7BJ/wVp4T/DwPwXnxaJ5GOSH9sOGBjc4GGTZgRpYchhYGbEo0XixuNj1oxtyQaSPWlmkr3bJOpBfjnYcy4dpxb+/sTmmz/b7Az42Q8/k/i5zcZYvv/wwwc/yqxxagEBZh4kW8HkAbzqgYDxByEVo2AUjIJRMLIBAA5FT969PEgUAAAAAElFTkSuQmCC","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Cavagnero","suffix":""}],"badges":[],"createdAt":"2023-03-25 03:29:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2734168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2734168/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-61274-1","type":"published","date":"2024-05-29T13:02:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":35738514,"identity":"c70b24b7-91a6-42c4-9e7b-158dc7bd4efe","added_by":"auto","created_at":"2023-04-13 23:21:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCartoons illustrating model proteins and constructs employed in this work. a) \u0026nbsp;\u003c/strong\u003eStructure of \u003cem\u003eE. coli\u003c/em\u003e flavohemoglobin (Hmp), the model foldable protein used in this study. Hmp has three domains (shown in red, purple and blue). PDB code: 1GVH. \u003cstrong\u003eb)\u003c/strong\u003e \u0026nbsp;Cartoon illustrating the phosphorylated insulin receptor interacting region of the Grb14 protein from rat (PIR). PIR is the model intrinsically disordered protein used in this work. \u0026nbsp;\u003cstrong\u003ec)\u003c/strong\u003e \u0026nbsp;Schematic representation of the length and sequence location of the three domains of Hmp. The specific RNC constructs of the apo form of Hmp used in this work (denoted here as apoHmp) are also shown, with the respective chain lengths listed as subscripts. \u0026nbsp;\u003cstrong\u003ed)\u003c/strong\u003e \u0026nbsp;Linear bar illustrating the 91-residues length of PIR and its respective RNC.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/6c7cea24fba58cbd6126ceda.png"},{"id":35739476,"identity":"03d68c7c-136f-4fc1-bdab-901fc9096068","added_by":"auto","created_at":"2023-04-13 23:29:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrosslinking patterns of apoHmp\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1-140 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eand apoHmp\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1-189 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eRNCs and identification of interacting ribosomal proteins. \u0026nbsp;a)\u0026nbsp; \u003c/strong\u003eSDS-PAGE and Western blot data identifying r-proteins interacting with apoHmp\u003csub\u003e1-155\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs in the presence of the EDC crosslinker and the ribosome-release agent puromycin. Data show that the L23 r-protein interacts with apoHmp\u003csub\u003e1-55\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs. Corresponding data employing antibodies against L24 and L29 r-proteins, showing no interactions, are available in the SI. Here, RNC’s are denoted as nascent chains bound to tRNA, in which the ribosome has been removed due to SDS and heat from gel analysis.\u0026nbsp; \u003cstrong\u003eb) \u0026nbsp;\u003c/strong\u003eSDS-PAGE and Western blot data identifying r-proteins interacting with apoHmp\u003csub\u003e1-140\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs in the absence and presence of the EDC crosslinker and the ribosome-release agent puromycin. Data show that the L23 r-protein interacts with apoHmp\u003csub\u003e1-140\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs. Corresponding data employing antibodies against L24 and L29 r-proteins, showing no interactions, are available in the Supplementary Information. \u0026nbsp;\u003cstrong\u003ec) \u003c/strong\u003e\u0026nbsp;Low-pH SDS-PAGE analysis of apoHmp\u003csub\u003e1-189\u003c/sub\u003e RNCs in the absence and presence of the EDC crosslinker, TF chaperone and the RNC ribosome-release agent puromycin.\u0026nbsp; \u003cstrong\u003ed)\u003c/strong\u003e \u0026nbsp;Side-by-side SDS-PAGE and Western blot data identifying the interaction network of apoHmp\u003csub\u003e1-189\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs in the absence and presence of EDC, puromycin and TF chaperone. The L23 r-protein is found to interact with apoHmp\u003csub\u003e1-189\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eRNCs. Corresponding data employing antibodies against L24 and L29 r-proteins, showing no interactions, are available in the Supplementary Information. Uncropped gel images for all panels are shown in Figures S10 and S11. \u003cem\u003eE. coli \u003c/em\u003ecell strains are listed above each SDS-Page gel. Note that the wild-type (WT) cell-free system, corresponding to the WT strain, contains the following chaperone concentrations: TF (2-15 nM), DnaK (0.5 mM), DnaJ (0.04 mM) and GrpE (0.05 mM).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/419c54f548c83f8d1e3767ef.png"},{"id":35739809,"identity":"83991892-f728-49ce-81de-7afb152f4920","added_by":"auto","created_at":"2023-04-13 23:37:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":302808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of the 70S \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ribosome.\u0026nbsp; a) \u0026nbsp;\u003c/strong\u003eTop view of 50S subunit of the \u003cem\u003eE. coli\u003c/em\u003e ribosome highlighting the ribosomal proteins (r-proteins). This figure has been modified from (51) under a Creative Commons Attribution 4.0 International license. \u0026nbsp;\u003cstrong\u003eb) \u0026nbsp;\u003c/strong\u003eTop view of the 50S subunit of the \u003cem\u003eE. coli \u003c/em\u003eribosome displaying the electrostatic surface potential map and highlighting relevant r-proteins. Figure has been modified from (51) under a Creative Commons Attribution 4.0 International license.\u003cem\u003e \u003c/em\u003e\u0026nbsp;\u003cstrong\u003ec) \u0026nbsp;\u003c/strong\u003eTop view of 50S \u003cem\u003eE. coli\u003c/em\u003e ribosome highlighting r-protein charged and nonpolar residues. Figure has been modified from (51) a Creative Commons Attribution 4.0 International license. \u0026nbsp;\u003cstrong\u003ed) \u0026nbsp;\u003c/strong\u003eSide view of r-proteins near the vestibule of the ribosomal exit tunnel. Figure has been modified from\u003cem\u003e \u003c/em\u003e(51) \u003ca href=\"https://creativecommons.org/\"\u003eunder a Creative Commons Attribution 4.0 International license.\u003c/a\u003e\u0026nbsp; \u003cstrong\u003ee)\u0026nbsp; \u003c/strong\u003eTop view of \u003cem\u003eE. coli\u003c/em\u003e 50S ribosomal subunit highlighting the r-proteins that either interact (green) or do not interact (red)\u0026nbsp; with apoHmp\u003csub\u003e1-55\u003c/sub\u003e, apoHmp\u003csub\u003e1-140\u003c/sub\u003e and apoHmp\u003csub\u003e1-189 \u003c/sub\u003eRNCs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/37d19910029c33498244a341.png"},{"id":35738518,"identity":"4fb11bb1-3cb7-42b3-8f8c-e37ddf08b39d","added_by":"auto","created_at":"2023-04-13 23:21:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence-anisotropy decays reveal that apoHmp nascent chains have a compact N-terminal region.\u0026nbsp; \u003c/strong\u003eRepresentative frequency-domain fluorescence anisotropy decay data of \u0026nbsp;\u003cstrong\u003ea) \u003c/strong\u003e\u0026nbsp;apoHmp\u003csub\u003e1-140 \u003c/sub\u003eand \u0026nbsp;\u003cstrong\u003eb) \u003c/strong\u003e\u0026nbsp;apoHmp\u003csub\u003e1-189 \u003c/sub\u003eRNCs. \u0026nbsp;\u003cstrong\u003ec) \u003c/strong\u003e\u0026nbsp;Table summarizing anisotropy decay parameters including rotational correlation times (rc), order parameters (S) and cone semi-angles (8). The S,I, and F subscripts denote slow-, intermediate-, and fast-timescale motions, respectively. Uncertainties are reported as ± SE for n=3-5. Three-component anisotropy fits were selected as best fits if they led to a 2.5-fold (or larger) decrease in reduced , relative to two-component fits. The \u0026nbsp;of the chosen model is shown in bold. \u0026nbsp;\u003cstrong\u003ed) \u003c/strong\u003e\u0026nbsp;Table summarizing the number of amino acids comprising the RNC compact region, deduced from the rc, I rotational correlation time and assuming spherical, oblate ellipsoid, or prolate ellipsoid nascent-chain shapes. The parameter p denotes the axial ratio. \u0026nbsp;\u003cstrong\u003ee)\u003c/strong\u003e \u0026nbsp;Cartoon representation of apoHmp\u003csub\u003e1-140 \u003c/sub\u003eand apoHmp\u003csub\u003e1-189 \u003c/sub\u003eRNCs based on the fluorescence anisotropy decay data shown in this figure and the data in Fig. 2. The wild-type (WT) cell-free system, corresponding to the WT strain, was employed for the data shown in this Figure. This cell-free system contains the following chaperone concentrations: TF (2-15 nM), DnaK (0.5 mM), DnaJ (0.04 mM) and GrpE (0.05 mM).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/256d722a106a65d7c3da3284.png"},{"id":35738516,"identity":"9fb67166-9e68-456a-90d6-e4f8d22dbd43","added_by":"auto","created_at":"2023-04-13 23:21:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNascent chains of the foldable protein apoHmp interact with specific ribosomal proteins in a chain-lengh-dependent fashion. \u003c/strong\u003eCartoon illustrating the fact that foldable nascent chains of ApoHmp interact with r-protein L23 at short (55 residues) and medium-size (140 residues) chain lengths. As translation continues and nascent proteins get longer (189 residues), the interaction network extends to one additional r-protein, while preserving the approximate size of the n-terminal non-interacting compact region. This cartoon is based on data in Figs. 2 and 4.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/0880fa9821b548eb6e4cd80c.png"},{"id":35739477,"identity":"be0ca58d-8d79-4d5e-93cb-3cde2e86d0dc","added_by":"auto","created_at":"2023-04-13 23:29:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":160671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDC-crosslinking-detected urea titrations showing the apparent stability of RNC/-r-protein complexes.\u0026nbsp; a) \u0026nbsp;\u003c/strong\u003eScheme showing the expected effect of urea addition on RNCs and \u0026nbsp;\u003cstrong\u003eb) \u003c/strong\u003e\u0026nbsp;corresponding low-pH SDS-PAGE gels. \u0026nbsp;\u003cstrong\u003ec) \u0026nbsp;\u003c/strong\u003eFour RNCs were tested in these experiments:apoHmp\u003csub\u003e1-55\u003c/sub\u003e, apoHmp\u003csub\u003e1-140\u003c/sub\u003e, apoHmp\u003csub\u003e1-189\u003c/sub\u003e or PIR\u003csub\u003e1-91\u003c/sub\u003e. Note that PIR\u003csub\u003e1-91\u003c/sub\u003e is an intrinsically disordered protein (IDP).\u0026nbsp; \u003cstrong\u003ed) \u0026nbsp;\u003c/strong\u003eRepresentative SDS-PAGE analysis. Gel bands are reporters of the apparent stability of complexes between RNCs and either L23 or TF. Uncropped gel images are shown in Fig. S10. \u003cstrong\u003ee) \u0026nbsp;\u003c/strong\u003eRepresentative urea titrations of apoHmp\u003csub\u003e1-55\u003c/sub\u003e, apoHmp\u003csub\u003e1-140\u003c/sub\u003e, apoHmp\u003csub\u003e1-189\u003c/sub\u003e and PIR RNCs.\u0026nbsp; \u003cstrong\u003ef) \u0026nbsp;\u003c/strong\u003eΔG°\u003csub\u003eapp,unfold\u003c/sub\u003e values in the presence of low concentrations of chaperones (WT cell-free system concentrations: 2-15 nM of TF and 0.5 µM, 0.04µM and 0.05µM of and DnaK, DnaJ and GrpE respectively). Uncertainties are reported as ± SE for n=2-3. \u0026nbsp;\u003cstrong\u003eg) \u0026nbsp;\u003c/strong\u003eP-value table for a two- tailed Student’s T-test, comparing the ΔG°\u003csub\u003eapp,unfold\u0026nbsp;\u003c/sub\u003evalues of RNC/r-protein complexes. Green and orange boxes denote statistically different and statistically equivalent data, respectively, according to a 95% confidence interval. \u0026nbsp;\u003cstrong\u003eh) \u0026nbsp;\u003c/strong\u003eTable displaying relevant ΔG°\u003csub\u003eapp,unfold \u003c/sub\u003eand m-values . \u003cstrong\u003ei) \u003c/strong\u003eNECNOP plot (100) displaying net charge/residue as a function of hydrophobicity/residue of PIR\u003csub\u003e1-91\u003c/sub\u003e, apoHmp\u003csub\u003e1-55\u003c/sub\u003e, apoHmp\u003csub\u003e1-140\u003c/sub\u003e, apoHmp\u003csub\u003e1-189 \u003c/sub\u003eprotein chains. \u0026nbsp;\u003cstrong\u003ej) \u0026nbsp;\u003c/strong\u003eEstimated total solvent-accessible surface areas\u0026nbsp;of protein chains, assuming fully extended conformations. Values were computed with Surfracer (101).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/0c64dab832f0f5af55463aa1.png"},{"id":35738521,"identity":"e8de0265-2715-4d34-902c-312e1ec3275b","added_by":"auto","created_at":"2023-04-13 23:21:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow pH SDS-PAGE and urea-titration analysis of apoHmp1-189 in the absence and presence of TF and K/J/E chaperones.\u0026nbsp; a)\u0026nbsp; \u003c/strong\u003eCartoon illustrating the tested RNCs. \u003cstrong\u003eb) \u0026nbsp;\u003c/strong\u003eLow pH SDS-PAGE analysis of complexes including apoHmp\u003csub\u003e1-189 \u003c/sub\u003eRNC and either r-proteins or molecular chaperones as a function of increasing urea concentration. Uncropped gel images are shown in Figure S10. \u003cstrong\u003ec) \u0026nbsp;\u003c/strong\u003eRepresentative urea titration curves. \u0026nbsp;\u003cstrong\u003ed)\u0026nbsp; \u003c/strong\u003eΔG°\u003csub\u003eapp,unfold \u003c/sub\u003evalues in the absence and presence of low concentrations of molecular chaperones (WT cell-free system: 2-15 nM TF, and 0.5 µM, 0.04µM and 0.05µM K/J/E, respectively). Error bars denote standard error based on 2-3 experiments. \u0026nbsp;\u003cstrong\u003ee)\u0026nbsp; \u003c/strong\u003eFraction of RNC/r-protein complexes relative to total RNCs. Uncertainties are reported as ± SE for n=2-3.\u0026nbsp; \u003cstrong\u003ef)\u0026nbsp; \u003c/strong\u003eP-value table for two-tailed Student’s test assuming unequal variances, comparing ΔG°app,unfold values. Green and orange boxes denote statistically different and statistically equivalent data, respectively, according to a 95% confidence interval.\u0026nbsp; \u003cstrong\u003eg)\u0026nbsp; \u003c/strong\u003eP\u003cem\u003e-\u003c/em\u003evalue table comparing fractions of RNC/r-protein complexes. Statistical assessments were similar to those listed in panel f.\u0026nbsp; \u003cstrong\u003eh)\u0026nbsp; \u003c/strong\u003eTable showing ΔG°\u003csub\u003eapp,unfold\u003c/sub\u003e and m values of relevant complexes.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/568d6cca166e8379aa708c25.png"},{"id":35739808,"identity":"71c04361-4e1b-4502-ad19-ad7f3e6e3be3","added_by":"auto","created_at":"2023-04-13 23:37:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":129998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe TF molecular chaperone displaces interactions between RNCs and ribosomal proteins. \u0026nbsp;a) \u003c/strong\u003e\u0026nbsp;Low-pH SDS-PAGE gels showing interacting apoHmp\u003csub\u003e1-55\u003c/sub\u003e RNCs after addition of EDC at low (2-15 nM) and high (8 mM) TF concentrations.\u0026nbsp; \u003cstrong\u003eb)\u0026nbsp; \u003c/strong\u003ePictorial representation of interacting apoHmp\u003csub\u003e1-55 \u003c/sub\u003eRNCs and their respective populations (n=2 ± SE). \u0026nbsp;\u003cstrong\u003ec) \u003c/strong\u003e\u0026nbsp;Low-pH SDS-PAGE gels showing interacting apoHmp\u003csub\u003e1-140\u003c/sub\u003e RNCs after addition of EDC at low (2-15 nM) and high (8 mM) TF concentrations.\u0026nbsp; \u003cstrong\u003ed)\u0026nbsp; \u003c/strong\u003ePictorial representation of interacting apoHmp\u003csub\u003e1-140 \u003c/sub\u003eRNCs and their respective populations (n=2-3 ± SE).\u0026nbsp; \u003cstrong\u003ee) \u003c/strong\u003e\u0026nbsp;Low-pH SDS-PAGE gels showing interacting apoHmp\u003csub\u003e1-189\u003c/sub\u003e RNCs after addition of EDC at low (2-15 nM) and high (8 mM) TF concentrations.\u0026nbsp; \u003cstrong\u003ef)\u0026nbsp; \u003c/strong\u003ePictorial representation of interacting apoHmp\u003csub\u003e1-189 \u003c/sub\u003eRNCs and their respective populations (n= 2-4 ± SE). The WT cell-free systems have K/J/E chaperones at 0.5, 0.04 and 0.05 mM concentrations, respectively. Uncropped gel images are shown in Figs S10 and S11.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/a5a86bc20e63b2c7453924f7.png"},{"id":57544237,"identity":"92787a27-89f8-4c97-a299-b742b8f0a3c6","added_by":"auto","created_at":"2024-06-01 13:02:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2369688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/1a249f3f-3be9-4081-bb43-4c4330246f2e.pdf"},{"id":35738522,"identity":"a5936a98-ba72-4ca8-9968-070d30b3f9b4","added_by":"auto","created_at":"2023-04-13 23:21:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52481596,"visible":true,"origin":"","legend":"","description":"","filename":"MerribonetworksupSCIREP040923.docx","url":"https://assets-eu.researchsquare.com/files/rs-2734168/v1/328b6d9dbe010bd2e5ff6835.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRecent evidence suggests that the ribosome plays an active role in cotranslational protein folding and solubility \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. During translation, the nascent chain traverses the ribosomal exit tunnel, which is ca. 80\u0026ndash;100 \u0026Aring; long, 10\u0026ndash;35 \u0026Aring; wide \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and typically fits 30 to 40 nascent residues. \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Within the ribosomal exit tunnel and its nearby regions across the highly negatively charged outer surface of the ribosome\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, nascent chains encoding single-domain proteins become compact \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and acquire some secondary \u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and tertiary structure \u003csup\u003e5,28\u0026minus;32\u003c/sup\u003e. This set of observations proves the importance of the ribosome in nascent-protein structure formation.\u003c/p\u003e \u003cp\u003eDuring translation, the ribosome influences nascent protein chains at different levels. For instance, it renders nascent chains soluble relative to the corresponding ribosome-released proteins, thereby supporting cotranslational events devoid of undesirable aggregation \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Further, the inner geometry of the ribosomal exit tunnel favors formation of secondary nascent-chain structure, especially of α-helical \u003csup\u003e26,33\u0026minus;35\u003c/sup\u003e or β-sheet nature \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In addition, the ribosomal exit tunnel and vestibule enable acceleration of folding \u0026ndash; but not unfolding \u0026ndash; of a small single-domain protein, thereby stabilizing nascent protein chains relative to their free state in solution \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This effect was ascribed primarily to electrostatic interactions between nascent proteins and ribosome \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. On the other hand, the ribosome may also destabilize single-protein domains, in case the domain is far removed from the peptidyl transferase center \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Collectively, these results highlight the influence of the ribosome on nascent protein folding.\u003c/p\u003e \u003cp\u003eThe ribosome is also known to establish physical noncovalent interactions with some nascent chains. As summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, these interactions were identified in a variety of experimental studies and can be divided into three categories. Namely, \u003cem\u003e(i)\u003c/em\u003e interactions between the ribosome and nascent chains carrying an N-terminal signal sequence \u003csup\u003e18,38\u0026minus;42\u003c/sup\u003e, \u003cem\u003e(ii)\u003c/em\u003e interactions between the ribosome and nascent chains bearing a C-terminal ribosome-stalling or arrest sequence \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47 CR48 CR49\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003e(iii)\u003c/em\u003e interactions between the ribosome and nascent chains encoding intrinsically disordered proteins \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Additional studies are consistent with the presence of ribosome-nascent-chain interactions, though they do not directly prove their existence \u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, ribosome-bound nascent chains (RNCs) of intrinsically disordered character or bearing signal or arrest sequences are known to interact with ribosomal proteins. However, RNCs bearing foldable protein sequences and lacking linkers or signal/arrest tags have not been explicitly characterized in terms of experimentally detectable interactions with the ribosome.\u003c/p\u003e \u003cp\u003eFurther, little is known about how the nascent chain may affect certain components of the ribosome. For instance, empty 70S ribosomes are known to be more prone to dissociation than ribosomes bearing both mRNA and peptidyl tRNA. This conclusion was reached upon addition of either ribosome-dissociation factors \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e or Hofmeister cosolutes \u003csup\u003e\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Other researchers established a similar finding upon depletion of magnesium ions \u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In a different study, addition of Hofmeister salts were employed to show that translation initiation complexes (including 70S in complex with initiator tRNA) disassemble more easily than peptidyl tRNAs bearing nascent chains \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Ribosomes carrying longer nascent chains were found to be less prone to dissociation \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Other studies examined the effect of magnesium ions and other Hofmeister ions on empty-70S-ribosome disassembly and how it changes sedimentation coefficients \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Yet, there is only a limited number of studies targeting the effect of non-Hofmeister denaturing agents on the ribosome. For instance, it is known that the 30S subunit disassembles in the presence of 6 M urea \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In addition, urea lowers the melting temperature and sedimentation coefficient of the 50S ribosomal subunit \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The 30S subunit is more sensitive to thermal denaturation than the 50S subunit, and the 70S ribosome is most thermally stable \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. In addition, 70S ribosomes bearing a nascent chain are less prone to chemical denaturation than empty ribosomes \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we address the lack of knowledge on nascent-chain/ribosome interactions by exploring them in the case of ribosome-bound nascent chains (RNCs) of increasing length belonging to a foldable protein sequence. We find that RNCs up to chain length 140 interact only with one ribosomal protein (r-protein), i.e., L23, in the vicinity of the ribosomal exit tunnel. This result is surprising because the ribosomal surface near the tunnel exit bears several r-proteins. A wider interaction network, including one additional ribosomal protein and the trigger factor (TF) chaperone, gets established as the nascent chain elongates up to 189 residues. The populations of RNC-interacting proteins evolve as a function of chain elongation. Specifically, interactions with r-proteins get partially or completely replaced by interactions with the TF molecular chaperone, as TF concentration increases up to physiologically relevant values. In order to gain additional insights on the potential stabilizing role of the interactions, we also investigate the effect of the above RNC/r-protein interactions on the bacterial ribosome. The apparent stability of the complexes between RNCs of foldable and intrinsically disordered protein sequences and specific r-proteins is weak and, surprisingly, does not vary significantly with RNC sequence, length, net charge and hydrophobicity. Hence, we propose that the ribosome provides unbiased thermodynamic assistance to nascent chains regardless of their electrostatic and nonpolar character. As an ancillary finding, we also show that the apparent thermodynamic stability of the peptidyl transferase center (PTC) and all ribosomal proteins is not affected by RNC-ribosome interactions. Further, short peptidyl-tRNAs (snc-tRNAs) stabilize the 70S ribosome against denaturation by the non-Hofmeister cosolute urea, suggesting a multi-step model for the disassembly of ribosome-RNC complexes.\u003c/p\u003e \u003cp\u003eIn all, our results highlight the supporting role of the ribosome for newly synthesized protein chains, showing that it establishes interactions with RNCs via specific r-proteins.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e \u003cb\u003eExperimental design.\u003c/b\u003e This work focuses on ribosome-bound nascent chains (RNCs) derived from \u003cem\u003eEscherichia coli\u003c/em\u003e flavohemoglobin (Hmp, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and from the phosphorylated insulin receptor interacting region (PIR) of the growth factor receptor-bound protein 14 from \u003cem\u003eRattus norvegicus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The Hmp protein comprises three domains, an N-terminal heme-binding (domain 1), a flavin adenine dinucleotide-binding (domain 2) and a C-terminal nicotinamide adenine dinucleotide-binding domain (domain 3), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Several RNC chain lengths were examined, and all pertinent constructs are shown as solid bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. Hmp plays a key role in O\u003csub\u003e2\u003c/sub\u003e, NO and CO transport in \u003cem\u003eE. coli\u003c/em\u003e, and is involved in a variety of signaling pathways \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The cofactor-free form of Hmp is denoted as apoHmpH. Importantly, previous studies established that the N-terminal globin domain of Hmp is stable and folded even in its apo form \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Our second target protein, PIR, is intrinsically disordered \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, i.e., an IDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The specific nascent-chain constructs of both proteins analyzed in this work are schematically illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRibosome-nascent-protein interactions were probed with via the well-characterized zero-length chemical crosslinker carbodiimide 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Nascent chains were chemically crosslinked via known procedures \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e involving a combination of low-pH SDS-PAGE \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e and Western blotting in the absence and presence of the trigger factor (TF) chaperone. Notably, EDC enables the detection of existing noncovalent interactions and it does not provide an accurate quantitation of interacting populations, as discussed at length by Guzman-Luna \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Yet, in the presence of appropriate controls, EDC is an extremely valuable tool to detect the existence of protein-protein interactions within the ribosome-nascent-chain complex. In addition, relative changes in the extent of the interactions, for any given ribosome-nascent-chain complex (RNC) as a function of environmental changes (e.g., variable urea or chaperone concentrations) were also qualitatively assessed. In general, due to the established presence of crosslinking-incompetent populations in RNC/r-protein complexes detected via EDC \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, interacting populations tend to be underestimated. Site-specific fluorescence labeling of nascent proteins at their N terminus enables focusing exclusively on interactions involving the nascent protein. Low-pH-gel and Western-blot were collected to explore interactions between nascent chains and r-proteins. It is worth noting that EDC does not have high accessibility within the exit-tunnel core \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Therefore, detection of interactions within the tunnel core is not expected, within our experimental setup.\u003c/p\u003e \u003cp\u003eIt is also important to mention that, under our experimental conditions, EDC does not report on interactions involving nascent protein chains and ribosomal RNA (rRNA). In the presence of imidazole, crosslinks between RNA 5' phosphate and aliphatic amines of proteins are known to take place \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. However, our samples did not contain imidazole, and this chemical would anyways be unable to detect interactions not involving the 5\u0026rsquo; end of RNA. Therefore, even in the presence of imidazole, EDC would likely underestimate all potential interactions with RNA. Thus, interactions between nascent proteins and rRNA are beyond the scope of this study.\u003c/p\u003e \u003cp\u003eThe compaction, tumbling rates, size (expressed in terms of approximate number of residues)and local-motion amplitude of non-interacting RNC regions were assessed via fluorescence depolarization in the frequency domain. In this way, it was possible to gain complementary and more comprehensive insights into RNC conformational characteristics. The apparent thermodynamic stability of r-proteins was collectively assessed by Trp fluorescence emission spectroscopy as a function of urea concentration. The apparent stability of the peptidyl transferase center (PTC) of the ribosome in the presence of a variety of RNCs was evaluated by urea titrations upon detection via a puromycin-release assay. Finally, the empty-ribosome and RNC assembly status of the ribosome, in terms of 30S, 50S and 70S subunits, was assessed via sucrose-gradients and negative-staining transmission electron microscopy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eApoHmp RNCs of increasing length interact with ribosomal protein L23.\u003c/b\u003e We elected to probe whether apoHmpH RNCs interact with the L23, L24 and L29 r-proteins, which reside within the vestibule of the ribosomal exit tunnel and the adjacent outer surface of the ribosome. We explored the interaction patterns of three representative nascent chains, namely apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e. The data for these RNCs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A side-by-side comparison between low-pH SDS-Page gels \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e and Western blots indicate that all three nascent proteins interact with ribosomal protein L23. Western blotting carried out with antibodies against ribosomal proteins L24 and L29, shown in the Supplementary Information (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicates no evidence for interactions between the L24 and L29 r-proteins and the target RNCs. Therefore, apoHmpH RNCs interact exclusively with ribosomal protein L23. In contrast, intrinsically disordered PIR\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;91\u003c/sub\u003e RNCs, analyzed in previous studies\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, interact with both the L23 and L29 ribosomal proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder our experimental conditions, the fraction of interacting RNCs is different, for nascent chains derived from apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c) and intrinsically disordered PIR\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;91\u003c/sub\u003e\u003csup\u003e51\u003c/sup\u003e. Indeed, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e and PIR\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;91\u003c/sub\u003e crosslink only in part, unlike apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs, which are nearly 100% crosslinked. On the other hand, the larger extent of crosslinking of the foldable apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs relative to apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e and PIR may be mainly a consequence of the greater number of EDC-reactive residues of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e (25 EDC-reactive residues, ca. 20 beyond the tunnel core) and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e (36 EDC-reactive residues, ca. 30 beyond the tunnel core) relative to apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e (11 EDC-reactive residues, ca. 5 beyond the tunnel core) and PIR (14 EDC-reactive residues, ca. 12 beyond the tunnel core). In support of this argument (see sections below), the urea sensitivity of the L23 / RNC complexes is similar for all RNCs, suggesting comparable interaction strengths.\u003c/p\u003e \u003cp\u003eImportantly, given that fluorescence anisotropy-decay data (see later sections) show that apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs have dynamic and independently tumbling N-terminal compact regions, it is clear that the RNC regions interacting with the ribosomal surface cannot include any significant fraction of N-terminal residues belonging to the compact region.\u003c/p\u003e \u003cp\u003eIn the case of the longest RNCs analyzed in this work, corresponding to apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e, we found an additional interacting complex of higher molecular weight, which we denote as RP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The corresponding population includes r-protein L23, according to Western blotting, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, and one additional unidentified protein of c.a. 6\u0026ndash;10 kDa, according to molecular weight arguments. Our Western blots indicate that L29 (7 kDa) is not present in the RP2 band (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef). Yet, other cytoplasmic \u003cem\u003eE. coli\u003c/em\u003e chaperones and ribosome interactors (GroEL, GroES, SecB, DnaK/DnaJ/GrpE, SRP and ClpB; MW range: 48\u0026ndash;80 kDa) are ruled out, as they would appear well above the RP2 complex in our gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). Due to its close spatial proximity to L23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b,c and d) and based on the above-mentioned molecular-weight arguments, it is possible that RP2 comprises both L23 and L29. However, our monoclonal antibodies against r-protein L29 were unable to capture an L29 epitope, as part of the crosslinked complex adsorbed onto the PVDF membrane. To further test for the possibility that L29 being part of the RP2 interacting protein pair, additional experiments in the presence of polyclonal antibodies against L29 will be carried out in the future.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe data in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and S2 also show that a fraction of the apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs interacts with the trigger factor (TF) chaperone. The presence of these contacts was assessed upon comparing denaturing gels for data collected with wild-type (WT) and TF-depleted (\u003cem\u003eDtig\u003c/em\u003e) \u003cem\u003eE. coli\u003c/em\u003e cell strains. Indeed, RNC / TF interactions are already known to exist from previous literature, especially for nascent proteins longer than ca. 100\u0026ndash;110 residues\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e 42,76\u0026minus;79\u003c/sup\u003e Previous studies also showed that TF interacts with client proteins that bear a fairly expanded conformation, in their bound state\u003csup\u003e\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOn the other hand, the small observed fraction of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e interacting with TF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) is unexpected. This result implies the presence of a highly stretched conformation of this short (55-residue) RNC, which must reside mostly within the 80\u0026ndash;100 \u0026Aring;-long ribosomal exit tunnel. Yet, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e manages to reach out to the TF chaperone, which is known to dock onto the outer surface of the ribosome via the L23 and L29 r-proteins. This conformational stretching experienced by a small fraction of the short apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e RNC is fascinating and unprecedented. Indeed, the presence of L23-docked and TF-docked apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e nascent-chains suggests that cotranslational conformational sampling can take place even in the case of a fairly short RNC.\u003c/p\u003e \u003cp\u003eTF and L23 are known to interact with one another on the ribosome \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e, though we presently cannot explicitly discriminate whether the nascent chains interact with L23 and TF, or if the nascent chain interacts with TF, which in turn interacts with L23. Here, we propose the simplest scenario namely that RNCs interact with TF only and, in turn, TF interacts with L23, which is known to be the TF docking site on the ribosome \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, in this work we only analyzed the behavior of RNCs in the presence of moderate concentrations of the 70 kDa Hsp70 chaperone. Hsp70 was studied in the context of the DnaK/DnaJ/GrpE chaperone system, denoted here as K/J/E. Now, the wild-type (WT) cell-free system used in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b,d contains K/J/E at 0.5, 0.04, 0.05 mM concentrations, respectively, which are significantly lower than physiologically relevant values. Interestingly, at these low K/J/E concentrations, none of these chaperones is bound to the apoHmpH\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e resuspended RNCs, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b,d. Therefore, the Hsp70 chaperone does not bind the RNCs studied in this work. The effect of higher, more physiologically relevant (20\u0026ndash;50 mM) K/J/E concentrations will be studied elsewhere \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn all, our data show that apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e, apoHmpH\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs interact with either the L23 r-protein alone (apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e and apoHmpH\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), with L23 and another ribosomal protein (apoHmpH\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e), or with the TF chaperone (all RNCs, including apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e). We propose that these two classes of interactions (i.e., with r-proteins and with TF) play a similar chaperone-like role. This concept is consistent with previous studies, which showed that the ribosome serves as a nascent-chain solubilizing agent even in the absence of chaperones \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The fairly solvent-exposed nonpolar patch of the L23 r-protein, highlighted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d, is also consistent L23 being able to interact with nonpolar regions of RNCs. Future work will focus on genomic \u003cem\u003eE. coli\u003c/em\u003e r-protein modifications aimed at disrupting the detected interactions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRibosome-bound apoHmp nascent chains of variable length have a compact N-terminal region.\u003c/b\u003e Next, we performed fluorescence depolarization decay experiments in the frequency domain \u003csup\u003e\u003cspan additionalcitationids=\"CR87\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e to probe the rotational dynamics of nascent chains encoding foldable sequences. This technique has been previously employed to assess the rotational correlation time (t\u003csub\u003ec\u003c/sub\u003e) and amplitude of rotational motions of RNCs \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. The goal of this experiment was to determine whether RNCs harboring long nascent chains display any degree of compaction. We focused on RNCs of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e, corresponding to the N-terminal domain 1 of Hmp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and RNCs of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e, which comprise Hmp\u0026rsquo;s domain 1 and an additional 49 C-terminal residues belonging to domain 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Nascent proteins were site-specifically labeled at their N terminus with the BODIPY-FL fluorophore as described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Once information on nascent-chain compaction is in hand, the interplay between ribosome and nascent-chain interactions, and their sensitivity to urea denaturation can be more rationally explored and understood, as apparent in the sections below.\u003c/p\u003e \u003cp\u003eRepresentative data for apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e are shown in panels a and b of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, respectively. Both RNCs display informative frequency-domain anisotropy decay profiles. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and consistent with the very low reduced c\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e values, the fits that include 3 rotational-tumbling components give the best results. Importantly, panels c and d of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show that both apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs are characterized by an N-terminal compact domain that tumbles independently from the ribosome. This conclusion was reached upon applying known procedures based on a combination of microscale viscosity and fluorescence depolarization in the frequency domain. In both cases, this domain spans ca. 63 to 94 residues, depending on the exact shape. Note that RNC shape assessment is beyond the scope of this work. Regardless of the actual overall morphology of the compact domains, the fact that a compact domain of identical size is observed for both apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e suggests that both constructs undergo a similar degree of partial folding on the ribosome. Surprisingly, the observed size of the compact domain of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs is significantly smaller than the size of the entire apoHmp domain 1, which comprises 140 residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Therefore, biosynthesis of the additional 49 C-terminal amino acids belonging to domain 2 is not sufficient to lead to complete folding of the N-terminal domain domain 1, for this protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, cone semi-angle analysis of the fluorescence anisotropy decay data (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) shows that the compact domain of Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e RNCs spans a slightly wider cone semi-angle (26.5⁰ \u0026plusmn; 0.5⁰) than Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e RNCs (20⁰ \u0026plusmn; 0.2⁰), consistent with the fact that the latter construct likely projects slightly further out from the ribosomal surface than the shorter Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e construct. In all, our fluorescence anisotropy data show that the Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e and Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e nascent chains are both comparably compact and no more than partially folded, while on the ribosome, with Hmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e spanning a slightly wider cone semi-angle.\u003c/p\u003e \u003cp\u003eAll the above information on fluorescence anisotropy decays is pictorially recapitulated by the cartoons of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. The images presented in this figure also show a variety of compact species that take into account the r-protein-interacting and non-interacting populations deduced from the SDS-Page gels and Western blotting data of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In order to recapitulate the RNC/r-protein interaction profiles and nascent-protein conformation knowledge gained so far, a model highlighting the leading trends is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The RNCs displayed in this figure highlight the evolution of foldable apoHmpH nascent-chain interactions with r-proteins as a function of chain elongation. Briefly, when the nascent chain reaches a 55-residue length, the main detected interactions are with r-protein L23. No compact region is shown at this chain length, consistent with known fluorescence anisotropy-decay data collected on RNCs of a related globin \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. As the nascent chain gets longer and reaches a length of 140 residues, interactions with L23 are still present, but the chain also features a non-interacting compact region that spans a cone semi-angle of ca. 20⁰. As the nascent chain reaches 189-residue length, two classes of RNC populations interacting with r-proteins are present. The former interacts only with L23 and the other one also interacts with an additional ribosomal protein. In both cases, an N-terminal compact region encompassing 65\u0026ndash;94 residues is also detected. Further chain elongation and ribosome-release processes, which are beyond the scope of this study, are expected to give rise to the full-length ribosome-released folded protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNascent chain-L23 complexes have the same apparent stability regardless of RNC sequence.\u003c/b\u003e To further explore the nature of the interactions between the L23 r-protein and nascent chains of increasing length and variable sequence, we performed urea titrations with chemical crosslinking detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b). EDC readily reacts with amines and carboxylic acid functional groups, and there is no loss of EDC reactivity even in the presence of high urea concentrations \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. It is worth noting that the interactions identified in this work are not induced by the covalently N-terminal-linked BODIPY-502 fluorophore, as previous work has shown that this fluorophore does not interact with resuspended ribosomes under conditions like those of the present study \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, by unfolding the complex in the presence of urea and subsequently adding EDC, we expect to gain insights into the urea sensitivity of nascent chain-L23 complexes. While different RNC constructs are expected to bear a different number of EDC-reactive residues, denaturant titration of RNC complexes always examine the same nascent chain at variable urea concentration. Therefore, it is not necessary to normalize the data on a per-EDC-reactive-residue basis, as done in other studies \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter collecting gel data on representative apoHmp and PIR nascent chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), we estimated the apparent stability (ΔG\u0026deg;\u003csub\u003eapp,unfold\u003c/sub\u003e) of nascent chain-L23 complexes following a known extrapolation method which is further described in the SI methods \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Representative EDC-mediated urea titrations are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed. Corresponding plots and apparent-stability data are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee,f. The matching two-tailed Student\u0026rsquo;s t-test is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh, the apparent stability values for the apoHmp and PIR nascent-chain/L23 complexes (RP1 complexes) range between DG\u003csup\u003e0\u003c/sup\u003e\u003csub\u003eapp,unf\u003c/sub\u003e of 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 and 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. As shown in the t-test of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, all complexes display the same apparent stability within error. The corresponding values for the apparent unfolding equilibrium constants K\u003csub\u003eapp\u003c/sub\u003e (Table S2) are within the 590\u0026thinsp;\u0026plusmn;\u0026thinsp;340 mM to 58\u0026thinsp;\u0026plusmn;\u0026thinsp;41 mM range. These values, if regarded as estimates of the lower limits of the expected dissociation constants of r-protein/RNC complexes, suggest that the binding affinity of the apoHmp and PIR nascent-chain/L23 complexes (RP1 complexes) is overall rather weak. This qualitative estimate is consistent with the need for the interactions to be continuously remodeled during translation elongation. Interestingly, the observed trends apply even though the nascent-chain portions emerging from the ribosomal exit-tunnel core have widely different nonpolar and net-charge-per-residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei) as well as widely different total nonpolar surface accessible surface-area values (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eIn summary, the urea-titrations in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e show that the urea sensitivity of r-protein-nascent-chain complexes is similar regardless of the nature and length of the nascent chain, across the short and long (55- to 189-residue) chains examined here. In other words, RNC/-r-protein complexes have the same apparent stability, even though the RNCs have widely different physical properties and compaction (as discussed above and) and in the case of PIR, lack of compaction (discussed in previous work). \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Given that the amino-acid sequences of the interacting regions of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e must be different yet the interactions are of comparable apparent strength, the contacts are likely to be of nonspecific nature (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-h). This scenario, again, is consistent with the fact RNC-r-protein interactions likely need continuous remodeling during translation elongation.\u003c/p\u003e \u003cp\u003eFinally, the urea titrations described in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e are highly informative, as they also display the urea dependence of complexes between nascent chains and the trigger factor chaperone (RNC/TF complexes), e.g., see selected upper bands in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed. The quality of the data for the RNC/TF complexes was rather poor due to unreliable pre-transition baselines, therefore we did not deduce apparent stability values. On the other hand, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and in the plots of Fig. S3, the complexes with the TF chaperones are consistently less stable than the corresponding complexes with the L23 protein. This result suggests that the interactions between RNCs and the TF chaperone are even weaker than the interactions between RNCs and the L23 r-protein. Hence, nascent chains interacting with TF may in general be allowed more extensive conformational sampling in their bound state than nascent chains interacting with r-proteins. Additional future work will be devoted to further explore this hypothesis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNascent chain and r-protein interaction strength does not vary in the presence of one or more molecular chaperone.\u003c/b\u003e Next, we explored the effect of molecular chaperones TF and Hsp70 on the RNC-r-protein interactions via the same type of EDC-mediated urea titrations employed in the last section. The effect of Hsp70 was examined in the context of the K/J/E chaperone system. TF is known to associate with prokaryotic ribosomes \u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e and K/J/E works in cooperation with TF \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e to promote nascent-protein folding and prevent nascent-protein aggregation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e,\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e,\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFirst, we evaluated apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e devoid of both TF and the Hsp70 chaperone system (K/J/E), apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e in the presence of low concentrations of TF (2\u0026ndash;15 nM) only, apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e in the presence of low concentrations of K/J/E (0.5, 0.04 and 0.05 mM, respectively) only, and apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e in the presence of both chaperones at low concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Urea titrations were carried out with increasing concentrations of urea (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), and the intensities of the crosslinked fractions were plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). We then obtained a ΔG\u0026deg;\u003csub\u003eapp, unfold\u003c/sub\u003e values for each of these constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed) and evaluated them with a two-tailed Student\u0026rsquo;s t-test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg), similarly to what done for the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Interestingly, the apparent strength of the L23-nascent chain complex was found to be statistically similar in all cases, regardless of chaperone concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, h). Given that this effect is not due to a variation in the fraction of crosslinked nascent chains to r-proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f), via Western Blot analysis, we conclude that the extent of interactions between nascent chains and L23 remains similar in the absence and presence of the TF and K/J/E chaperones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This finding suggests that nascent chains interact with ribosomal L23 in a structurally similar fashion regardless of the absence or presence of chaperones.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRNC/r-protein interactions are attenuated at high chaperone levels in a chain-length-dependent manner.\u003c/b\u003e To further elucidate the nature of RNC/chaperone complexes, we performed experiments at low (2\u0026ndash;15 nM) and high (8 \u0026micro;M) TF concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The high concentration values are representative of physiologically relevant TF concentrations, upon taking into account the differences in the concentrations of actively translating ribosomes in our cell-free system and in live \u003cem\u003eE. coli\u003c/em\u003e cells \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Interestingly, interactions with r-protein L23 are mostly displaced by interactions with TF, at high TF concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This effect, however, is more pronounced for longer RNCs, as shown by the representative gels of Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea,c,e, Fig. S2, and by the comprehensive analysis of the interacting populations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb,d,f. The shortest nascent chains of apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e only show c.a. 25% interactions with TF, even at high TF concentrations. We attribute this result to the fact that apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e is likely too short to form extensive interactions with the TF chaperone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo summarize, in the absence of TF (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and S2), the nascent chain either interacts primarily with r-proteins, mainly, L23. At higher, physiologically relevant concentrations of TF (8 \u0026micro;M TF), RNC interactions with r-proteins are displaced by interactions with this molecular chaperone. It is worth noting that TF is shared with thousands of additional cellular proteins \u003cem\u003ein vivo\u003c/em\u003e, unlike in the experiments shown here, which include purified resuspended RNCs. Further, our RNC concentrations are only 20\u0026ndash;30 nM. Hence, the TF chaperone is in large excess over RNCs even at the low chaperone concentrations employed here. This scenario differs from the cellular environment where both RNCs and molecular chaperones are at comparable concentrations, within the low uM range. Therefore, we propose that the actual cellular milieu likely involves RNC populations that interact in part with TF and in part with r-proteins.\u003c/p\u003e \u003cp\u003eIn all, our findings highlight the prominent role of the ribosome as an RNC interactor and suggest that the ribosome may have played a primordial chaperone role in Nature, before the evolution of the TF molecular chaperone.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe presence of very short nascent chains stabilizes the 70S ribosomal complex.\u003c/b\u003e After exploring nascent chain and r-protein interactions, we investigated the potential effect of these contacts on the bacterial ribosome. We began by performing a series of qualitative sucrose-gradient studies on \u003cem\u003eE. coli\u003c/em\u003e empty ribosomes and nascent-chain-loaded ribosomes. Our results, detailed in Fig. S4 and S5, showed that empty-70S ribosomes are more sensitive to urea denaturation than ribosomes bearing tRNAs linked to longer nascent chains. These results agree with previous sucrose gradient studies on RNCs \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. It appears that the snc-tRNA is responsible for most of the stabilizing effect (Figs. S4 and S5). Interestingly, these data suggest that length and amino-acid sequence of the nascent protein does not influence the urea sensitivity of ribosome-RNC complexes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe peptidyl transferase center site is largely unaffected by nascent-chain sequence and length, beyond 32 residues.\u003c/b\u003e Next, we probed whether nascent chains of different length, amino-acid sequence and foldability affect the apparent stability of specific regions of the ribosome. We directed our focus on the peptidyl transferase center (PTC) of the \u003cem\u003eE. coli\u003c/em\u003e ribosome, and we explored its urea sensitivity via a nascent-chain ribosome-release assay mediated by puromycin. These experiments employed a larger set of RNCs than in the previous sections.\u003c/p\u003e \u003cp\u003eThe results of puromycin-release-detected urea titrations are shown in Fig. S8 and further described in the Supplementary Information. Overall, the data show that the apparent stability of the ribosomal PTC is not affected by the presence of nascent chains longer than 32 residues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe global urea sensitivity of ribosomal proteins is largely unaffected by nascent-chain sequence and length.\u003c/b\u003e Next, we explored the effect of nascent-chain properties on the overall apparent stability of r-proteins via urea titrations based on Trp fluorescence emission. Trp is a well-known fluorescent reporter, and its emission properties are highly environmentally sensitive. Urea titrations were carried out and Trp fluorescence emission was monitored (Fig. S9b,c). Spectral shifts were regarded as reporters of r-protein folding, and centers of mass of emission spectra were assessed to generate titration curves reporting on the urea sensitivity of r-proteins. Note that incubation time totaling the measurements from beginning and end of experiments did not change the spectral center of mass (Fig. S9d). Urea titration data were processed according to Santoro and Bolen \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e,\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. Individual representative titration curves are shown in Fig. S9e.\u003c/p\u003e \u003cp\u003eThe ΔG\u0026deg;\u003csub\u003eapp, unfold\u003c/sub\u003e for each construct are plotted in Fig. S9f and corresponding t-test values are tabulated in Fig. S9g. Nearly all the constructs show statistically similar results, with ΔG\u0026deg;\u003csub\u003eapp, unfold\u003c/sub\u003e values ranging from 2 to 5 kcal\u0026bull;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Hence, the presence of peptidyl tRNA, regardless of nascent-chain characteristics, does not affect the urea sensitivity of r-proteins. As shown in previous sections, some nascent chains interact with the specific ribosomal protein L23. On the other hand, these interactions are not sufficiently strong to be detected via this assay, which monitors the overall sensitivity to urea of all r-proteins.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions.\u003c/b\u003e The presence of interactions between nascent chains bearing a foldable amino-acid sequence (with no signal or arrest tags) and specific ribosomal proteins has been suggested but never experimentally demonstrated, to date. Here, we identify the ribosomal protein L23 as a specific nascent-chain-interacting partner. L23 establishes noncovalent contacts with nascent chains of the multi-domain foldable model protein apoHmp, which lacks signal/arrest sequences. As nascent chains elongate, the RNC interaction network expands to another ribosomal protein. A non-interacting N-terminal compact RNC region comprising 63\u0026ndash;95 residues has also been identified for nascent chains bearing both 140 and 189 residues. A model recapitulating the presence of both RNC/r-protein interactions and non-interacting N-terminal regions is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Interactions with the TF take over, at high TF chaperone concentrations. Interestingly, ribosomal-protein/nascent-chain complexes have a similar weak apparent stability regardless of nascent-chain sequence, length and degree of foldability. Therefore, we propose that r-proteins shield nascent foldable proteins from aggregation before intramolecular folding becomes thermodynamically favorable, during and(or) immediately after translation. These findings are significant because they unveil the presence of interactions between a foldable nascent chain and the L23 ribosomal protein. In addition, the data reveal that these interactions coexist with nascent-chain compaction across the N-terminal region, suggesting ribosome-facilitated aggregation-prevention and conformational sampling.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cb\u003ePreparation of empty ribosomes.\u003c/b\u003e Empty ribosomes were generated from an in-house\u003cb\u003e-\u003c/b\u003eprepared A19 WT or A19 Δtig \u003cem\u003eE. coli\u003c/em\u003e S30 cell extract as described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were grown in Luria-Bertani (LB) broth and harvested at mid-log phase (A\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.6). The cells were lysed through a French press (thermo Electron Corporation, Waltham, MA) at ~\u0026thinsp;12,000 psi with a single passage. The lysate was subject to centrifugation at 30910 g and 20\u0026deg;C for 20 min. After centrifugation, the supernatant was incubated in translation buffer (0.75 M Tris-HCl pH 8.2, 7.5 mM DTT, 21 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 500 \u0026micro;M amino acids, 6 mM ATP, 67 mM PEP and 160 \u0026micro;g \u0026bull;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e pyruvate kinase) for 80 min to remove any endogenous mRNA from ribosomes. The supernatant was then dialyzed (12\u0026ndash;14 kDa MWCO) in buffer (10 mM Tris-HCl pH 8.2, 14 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 60 mM KOAc and 1 mM DTT) for 12 hrs, with a buffer exchange every 4 hours. The resulting A19 cell extract was used as the empty-ribosome sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of RNCs.\u003c/b\u003e RNCs were generated using an in-house prepared A19 \u003cem\u003eE. coli\u003c/em\u003e transcription-translation coupled cell-free system \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e as described. Cell strains either including (WT) or lacking (\u003cem\u003eΔtig\u003c/em\u003e) the trigger factor gene were employed \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Hsp70 chaperone activity was suppressed via the KLR-70 peptide \u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e to a final concentration of 0.2 mM. Transcription-translation proceeded for 30 min at 37\u0026deg;C in the presence of BODIPY-FL-Met-tRNA\u003csup\u003ef\u0026minus;Met\u003c/sup\u003e to specifically label RNCs at the N terminus. BODIPY-FL-Met-tRNA\u003csup\u003ef\u0026minus;Met\u003c/sup\u003e was prepared as described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. RNCs were stalled at various lengths to generate the desired apoHmp and PIR constructs via oligodeoxynucleotide-directed mRNA cleavage \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e,\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. An anti-ssrA oligonucleotide \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e was added to a final concentration of 12.83 pmol \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to prevent premature release of stalled RNCs. RNC pellets were isolated via a sucrose cushion (1.1 M sucrose, 20 mM tris base, 10 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 500 mM NH\u003csub\u003e4\u003c/sub\u003eCl, and 0.5 mM EDTA, 1 mM DTT, pH 7.0, as described)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and subjected to ultracentrifugation at 160,000 g for 1 hr at 4\u0026deg;C. The purified pellet was dissolved in resuspension buffer (10 mM tris-HCl, 10 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 60 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 0,5 mM EDTA and 1.0 mM DTT, pH 7.0) by shaking in an orbital shaker at 200 rpm on ice for 1 hr.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics and reproducibility\u003c/b\u003e. Statistical data analysis was performed with Excel V. 16.70 software. Data are displayed as the mean with \u0026plusmn; the standard error (SE), with the number of independent experiments listed in parenthesis, (e.g., n\u0026thinsp;=\u0026thinsp;2). Statistically meaningful differences between sets of data were determined via the two-tailed Student t-test. Pairs of results were regarded as statistically different if bearing \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOther experimental procedures.\u003c/b\u003e Experimental details on frequency-domain fluorescence-anisotropy, sucrose gradients, low-pH gels, puromycin assays, chemical crosslinking and urea titrations are available in the Supplementary Information.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of the study are available from the corresponding author, S.C., upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to M. Dalphin for helpful discussions. This work was funded by the National Science Foundation (NSF) grants MCB-1616459 and MCB-0951209 (to S.C). M. M. M. and R.B.H. received NIH TEAM-Science Fellowships from the University of Wisconsin-Madison and M.M.M received the Straka Fellowship from the University of Wisconsin-Madison. A. E. V. received a National Science Foundation GRFP graduate fellowship and a Science and Medicine Graduate Research Scholars Fellowship from the University of Wisconsin-Madison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M. designed and performed experiments, analyzed the data including statistical analysis, and wrote the manuscript. V.G.L. performed Western blotting experiments and matching data analysis. A.V. contributed to method development and participated in the writing of early versions of the manuscript. R.H. performed fluorescence depolarization experiments and took care of matching data analysis. A.S. participated in method development. W.W. contributed to figure preparation and design and helped with urea titration experiments. S.C. designed the project, participated in data analysis, wrote the manuscript and contributed to manuscript editing. A.F. performed some of the Western blotting experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWilson, D. N. \u0026amp; Beckmann, R. The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling. Curr. Opin. Struct. Biol. 21, 274\u0026ndash;282, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKramer, G., Boehringer, D., Ban, N. \u0026amp; Bukau, B. The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat. Struct. Mol. Biol. 16, 589, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFedyukina, D. V. \u0026amp; Cavagnero, S. Protein Folding at the Exit Tunnel. Annu. Rev. Biophys. 40, 337\u0026ndash;359, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechmann, S., Willmund, F. \u0026amp; Frydman, J. The ribosome as a hub for protein quality control. Mol. Cell 49, 411\u0026ndash;421, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiutkute, M., Samatova, E. \u0026amp; Rodnina, M. V. Cotranslational folding of proteins on the ribosome. Biomolecules 10, 97, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaudby, C. A., Dobson, C. M. \u0026amp; Christodoulou, J. Nature and Regulation of Protein Folding on the Ribosome. Trends Biochem. Sci 44, 914\u0026ndash;926, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAddabbo, R. M. \u003cem\u003eet al.\u003c/em\u003e Complementary Role of Co- and Post-Translational Events in De Novo Protein Biogenesis. J. Phys. Chem. B 124, 6488\u0026ndash;6507, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMecha, M. F., Hutchinson, R. B., Lee, J. H. \u0026amp; Cavagnero, S. Protein folding in vitro and in the cell: From a solitary journey to a team effort. Biophys. Chem. 287, 106821, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBan, N., Nissen, P., Hansen, J., Moore, P. B. \u0026amp; Steitz, T. A. The complete atomic structure of the large ribosomal subunit at 2.4 \u0026Aring; resolution. Science 289, 905\u0026ndash;920, (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarms, J. \u003cem\u003eet al.\u003c/em\u003e High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107, 679\u0026ndash;688, (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNissen, P., Hansen, J., Ban, N., Moore, P. B. \u0026amp; Steitz, T. A. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis. Science 289, 920\u0026ndash;930, (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoss, N., Gerstein, M., Steitz, T. \u0026amp; Moore, P. The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893\u0026ndash;906, (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalkin, L. I. \u0026amp; Rich, A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J. Mol. Biol. 26, 329\u0026ndash;346, (1967).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlobel, G. \u0026amp; Sabatini, D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions: I. Location of the polypeptides within ribosomes. J. Cell Biol. 45, 130\u0026ndash;145, (1970).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, S., Sakai, H. \u0026amp; Wiedmann, M. NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. J. Cell Biol. 130, 519\u0026ndash;528, (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKramer, G., Ramachandiran, V. \u0026amp; Hardesty, B. Cotranslational folding\u0026mdash;omnia mea mecum porto? The international journal of biochemistry \u0026amp; cell biology 33, 541\u0026ndash;553, (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsalkova, T., Odom, O., Kramer, G. \u0026amp; Hardesty, B. Different conformations of nascent peptides on ribosomes. J. Mol. Biol. 278, 713\u0026ndash;723, (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoolhead, C. A., McCormick, P. J. \u0026amp; Johnson, A. E. Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins. Cell 116, 725\u0026ndash;736, (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFedyukina, D. V., Jennaro, T. S. \u0026amp; Cavagnero, S. Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms. J. Biol. Chem. 289, 6740\u0026ndash;6750, (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis, J. P., Bakke, C. K., Kirchdoerfer, R. N., Jungbauer, L. M. \u0026amp; Cavagnero, S. Chain dynamics of nascent polypeptides emerging from the ribosome. ACS Chem. Biol. 3, 555\u0026ndash;566, (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHutchinson, R. B., Chen, X., Zhou, N. \u0026amp; Cavagnero, S. Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins. J. Phys. Chem. B 125, 6543\u0026ndash;6558, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis, J. P., Culviner, P. H. \u0026amp; Cavagnero, S. Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions. Protein Sci. 18, 2003\u0026ndash;2015, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, J. \u0026amp; Deutsch, C. Secondary Structure Formation of a Transmembrane Segment in Kv Channels. Biochemistry 44, 8230\u0026ndash;8243, (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMingarro, I., Nilsson, I., Whitley, P. \u0026amp; Von Heijne, G. Different conformations of nascent polypeptides during translocation across the ER membrane. BMC Cell Biol. 1, 3, (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhushan, S. \u003cem\u003eet al.\u003c/em\u003e α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 17, 313, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoolhead, C. A., McCormick, P. J. \u0026amp; Johnson, A. E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the dribosome and in their exposure to ribosomal proteins. Cell 116, 725\u0026ndash;736, (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgirrezabala, X. \u003cem\u003eet al.\u003c/em\u003e A switch from α-helical to β-strand conformation during co-translational protein folding. The EMBO Journal 41, e109175, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosolapov, A. \u0026amp; Deutsch, C. Tertiary interactions within the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 16, 405\u0026ndash;411, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNilsson, O. B. \u003cem\u003eet al.\u003c/em\u003e Cotranslational folding of spectrin domains via partially structured states. Nat. Struct. Mol. Biol. 24, 221\u0026ndash;225, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, P. \u003cem\u003eet al.\u003c/em\u003e Folding pathway of an Ig domain is conserved on and off the ribosome. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 115, E11284, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoltkamp, W. \u003cem\u003eet al.\u003c/em\u003e Cotranslational protein folding on the ribosome monitored in real time. Science 350, 1104\u0026ndash;1107, (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiutkute, M., Maiti, M., Samatova, E., Enderlein, J. \u0026amp; Rodnina, M. V. Gradual compaction of the nascent peptide during cotranslational folding on the ribosome. Elife 9, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiv, G., Haran, G. \u0026amp; Thirumalai, D. Ribosome exit tunnel can entropically stabilize α-helices. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 102, 18956, (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarino, J., von Heijne, G. \u0026amp; Beckmann, R. Small protein domains fold inside the ribosome exit tunnel. FEBS Lett. 590, 655\u0026ndash;660, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBa\u0026ntilde;\u0026oacute;-Polo, M. \u003cem\u003eet al.\u003c/em\u003e Transmembrane but not soluble helices fold inside the ribosome tunnel. Nat. Commun. 9, 5246, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWruck, F. \u003cem\u003eet al.\u003c/em\u003e The ribosome modulates folding inside the ribosomal exit tunnel. Commun. Biol. 4, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamelson, A. J., Jensen, M. K., Soto, R. A., Cate, J. H. D. \u0026amp; Marqusee, S. Quantitative determination of ribosome nascent chain stability. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 113, 13402\u0026ndash;13407, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhushan, S. \u003cem\u003eet al.\u003c/em\u003e Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide. Mol. Cell 40, 138\u0026ndash;146, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisner, G., Moser, M., Sch\u0026auml;fer, U., Beck, K. \u0026amp; M\u0026uuml;ller, M. Alternate Recruitment of Signal Recognition Particle and Trigger Factor to the Signal Sequence of a Growing Nascent Polypeptide. J. Biol. Chem. 281, 7172\u0026ndash;7179, (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHouben, E. N. G., Zarivach, R., Oudega, B. \u0026amp; Luirink, J. \u003cem\u003eEarly encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome\u003c/em\u003e. Vol.\u0026nbsp;170 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeterson, J. H., Woolhead, C. A. \u0026amp; Bernstein, H. D. The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding. Mol. Microbiol. 78, 203\u0026ndash;217, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUllers, R. S. \u003cem\u003eet al.\u003c/em\u003e Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome. J. Cell Biol. 161, 679\u0026ndash;684, (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhushan, S. \u003cem\u003eet al.\u003c/em\u003e SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center. PLoS Biol. 9, 10, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCruz-Vera, L. R., Rajagopal, S., Squires, C. \u0026amp; Yanofsky, C. Features of Ribosome-Peptidyl-tRNA Interactions Essential for Tryptophan Induction of tna Operon Expression. Mol. Cell 19, 333\u0026ndash;343, (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakatogawa, H. \u0026amp; Ito, K. The ribosomal exit tunnel functions as a discriminating gate. Cell 108, 629\u0026ndash;636, (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeidelt, B. \u003cem\u003eet al.\u003c/em\u003e Structural Insight into Nascent Polypeptide Chain\u0026ndash;Mediated Translational Stalling. Science 326, 1412\u0026ndash;1415, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarragher, B. \u003cem\u003eet al.\u003c/em\u003e Current outcomes when optimizing \u0026lsquo;standard\u0026rsquo;sample preparation for single-particle cryo‐EM. J. Microsc. 276, 39\u0026ndash;45, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y., Wolfle, T. \u0026amp; Rospert, S. Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae. J. Biol. Chem. 288, 33697\u0026ndash;33707, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurridge, C. \u003cem\u003eet al.\u003c/em\u003e Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy. Chem. Sci. 12, 13120\u0026ndash;13126, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaudby, C. A., Burridge, C. \u0026amp; Christodoulou, J. Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex. J. Magn. Reson. 326, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzman-Luna, V., Fuchs, A. M., Allen, A. J., Staikos, A. \u0026amp; Cavagnero, S. An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel. Commun. Biol. 4, 1\u0026ndash;17, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnight, A. M. \u003cem\u003eet al.\u003c/em\u003e Electrostatic Effect of the Ribosomal Surface on Nascent Polypeptide Dynamics. ACS Chem. Biol. 8, 1195\u0026ndash;1204, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabrita, L. D. \u003cem\u003eet al.\u003c/em\u003e A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Nat. Struct. Mol. Biol. 23, 278\u0026ndash;285, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabrita, L. D., Hsu, S. T. D., Launay, H., Dobson, C. M. \u0026amp; Christodoulou, J. Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 106, 22239\u0026ndash;22244, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsu, S. T. D., Cabrita, L. D., Fucini, P., Christodoulou, J. \u0026amp; Dobson, C. M. Probing Side-Chain Dynamics of a Ribosome-Bound Nascent Chain Using Methyl NMR Spectroscopy. J. Am. Chem. Soc. 131, 8366-+, (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubramanian, A. R., Davis, B. D. \u0026amp; Beller, R. J. in \u003cem\u003eCold Spring Harbor symposia on quantitative biology\u003c/em\u003e. 223\u0026ndash;230 (Cold Spring Harbor Laboratory Press).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeller, R. J. \u0026amp; Davis, B. D. Selective dissociation of free ribosomes of Escherichia coli by sodium ions. J. Mol. Biol. 55, 477\u0026ndash;485, (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdelman, I. S., Ts'o, P. O. \u0026amp; Vinograd, J. The binding of magnesium to microsomal nucleoprotein and ribonucleic acid. Biochim. Biophys. Acta 43, 393\u0026ndash;403, (1960).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Der Saag, P. T., Vlak, J. M. \u0026amp; De Greef, T. F. Ribosomes from Xenopus laevis eggs and embryos in a cell-free protein-synthesizing system: translational regulation. Cell Differ. 4, 385\u0026ndash;397, (1976).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRon, E. Z., Kohler, R. E. \u0026amp; Davis, B. D. Magnesium ion dependence of free and polysomal ribosomes from Escherichia coli. J. Mol. Biol. 36, 83\u0026ndash;89, (1968).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOppenheim, J., Scheinbuks, J., Biava, C. \u0026amp; Marcus, L. Polyribosomes in Azotobacter vinelandii: I. Isolation, characterization and distribution of ribosomes, polyribosomes and subunits in logarithmically growing Azotobacter. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 161, 386\u0026ndash;401, (1968).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelley, W. S. \u0026amp; Schaechter, M. Magnesium ion-dependent dissociation of polysomes and free 70 s ribosomes in Bacillus megaterium. J. Mol. Biol. 42, 599\u0026ndash;602, (1969).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeller, R. J. \u0026amp; Lubsen, N. H. Effect of polypeptide chain length on dissociation of ribosomal complexes. Biochemistry 11, 3271\u0026ndash;3276, (1972).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpirin, A. S. Structural transformations of ribosomes (dissociation, unfolding and disassembly). FEBS Lett. 40, S28-S37, (1974).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpitnik-Elson, P., Greeman, B. \u0026amp; Abramovitz, R. The Influence of 6-M Urea on 30-S Ribosomes of Escherichia coli. Eur. J. Biochem. 49, 87\u0026ndash;92, (1974).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts, M. E. \u0026amp; Walker, I. O. Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 199, 184\u0026ndash;193, (1970).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts, M. E. \u0026amp; Walker, I. Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis 199, 184\u0026ndash;193, (1970).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIlari, A., Bonamore, A., Farina, A., Johnson, K. A. \u0026amp; Boffi, A. The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket. J. Biol. Chem. 277, 23725\u0026ndash;23732, (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonamore, A. \u0026amp; Boffi, A. Flavohemoglobin: Structure and reactivity. Iubmb Life 60, 19\u0026ndash;28, (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForrester, M. T. \u0026amp; Foster, M. W. Protection from nitrosative stress: A central role for microbial flavohemoglobin. Free Radic. Biol. Med. 52, 1620\u0026ndash;1633, (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEun, Y. J., Kurt, N., Sekhar, A. \u0026amp; Cavagnero, S. Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin. J. Mol. Biol. 376, 879\u0026ndash;897, (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoncoq, K. \u003cem\u003eet al.\u003c/em\u003e The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling. FEBS Lett. 554, 240\u0026ndash;246, (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHermanson, G. (Elsevier, 2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoare, D. t. \u0026amp; Koshland, D. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J. Biol. Chem. 242, 2447\u0026ndash;2453, (1967).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKirchdoerfer, R. N., Huang, J. J. T., Isola, M. K. \u0026amp; Cavagnero, S. Fluorescence-based analysis of aminoacyl- and peptidyl-tRNA by low-pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 364, 92\u0026ndash;94, (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaine, A., Lovmar, M., Wikberg, J. \u0026amp; Ehrenberg, M. n. Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences. J. Biol. Chem. 281, 28033\u0026ndash;28038, (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerbitz, L. \u003cem\u003eet al.\u003c/em\u003e Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590\u0026ndash;596, (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakshmipathy, S. K. \u003cem\u003eet al.\u003c/em\u003e Identification of nascent chain interaction sites on trigger factor. J. Biol. Chem. 282, 12186\u0026ndash;12193, (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh, E. \u003cem\u003eet al.\u003c/em\u003e Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo. Cell 147, 1295\u0026ndash;1308, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaio, T., Guan, X., Rossi, P., Economou, A. \u0026amp; Kalodimos, C. G. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344, 1250494, (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNilsson, O. B., M\u0026uuml;ller-Lucks, A., Kramer, G., Bukau, B. \u0026amp; von Heijne, G. Trigger factor reduces the force exerted on the nascent chain by a cotranslationally folding protein. Journal of molecular biology 428, 1356\u0026ndash;1364, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeckert, A. \u003cem\u003eet al.\u003c/em\u003e Structural characterization of the interaction of alpha-synuclein nascent chains with the ribosomal surface and trigger factor. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A\u003c/em\u003e 113, 5012\u0026ndash;5017, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eet al.\u003c/em\u003e Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A\u003c/em\u003e 113, 5012\u0026ndash;5017, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeuerling, E., Schulze-Specking, A., Tomoyasu, T., Mogk, A. \u0026amp; Bukau, B. Trigger factor and DnaK cooperate in folding of newly synthesized proteins. Nature 400, 693\u0026ndash;696, (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHesterkamp, T. \u0026amp; Bukau, B. Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli. The EMBO Journal 17, 4818\u0026ndash;4828, (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeechem, J. M. \u0026amp; Gratton, E. in \u003cem\u003eTime-Resolved Laser Spectroscopy in Biochemistry\u003c/em\u003e (ed J.R. Lakowicz) 70\u0026ndash;81 (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJameson, D. M., Gratton, E. \u0026amp; Hall, R. D. The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry. Appl. Spectrosc. Rev. 20, 55\u0026ndash;106, (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss, J. A. \u0026amp; Jameson, D. M. Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence. Photochem. Photobiol. Sci. 7, 1301\u0026ndash;1312, (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinreis, S. A., Ellis, J. P. \u0026amp; Cavagnero, S. Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome. Methods 52, 57\u0026ndash;73, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis, S. D. \u0026amp; Shafer, J. A. Conversion of exposed aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues. Biochimica et Biophysica Acta (BBA) - Protein Structure 303, 284\u0026ndash;291, (1973).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantoro, M. M. \u0026amp; Bolen, D. W. Unfolding free-energy changes determined by the linear extrapolation method.1.unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 8063\u0026ndash;8068, (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartl, F. U., Bracher, A. \u0026amp; Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324\u0026ndash;332, (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgashe, V. R. \u003cem\u003eet al.\u003c/em\u003e Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199\u0026ndash;209, (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeter, S. A. \u003cem\u003eet al.\u003c/em\u003e Polypeptide Flux through Bacterial Hsp70: DnaK Cooperates with Trigger Factor in Chaperoning Nascent Chains. Cell 97, 755\u0026ndash;765, (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWruck, F. \u003cem\u003eet al.\u003c/em\u003e Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches. J. Mol. Biol. 430, 438\u0026ndash;449, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePace, C. N. Measuring and increasing protein stability. Trends Biotechnol. 8, 93\u0026ndash;98, (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBakke, C. K., Jungbauer, L. M. \u0026amp; Cavagnero, S. In vitro expression and characterization of native apomyoglobin under low molecular crowding conditions. Protein Expr. Purif. 45, 381\u0026ndash;392, (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalphin, M. D., Stangl, A. J., Liu, Y. \u0026amp; Cavagnero, S. KLR-70: A Novel Cationic Inhibitor of the Bacterial Hsp70 Chaperone. Biochemistry 59, 1946\u0026ndash;1960, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehrmann, M. \u003cem\u003eet al.\u003c/em\u003e Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli. J. Biol. Chem. 273, 13898\u0026ndash;13904, (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonis-Keller, H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 7, 179\u0026ndash;192, (1979).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaeger-Weiss, S. K. \u003cem\u003eet al.\u003c/em\u003e Net charge and nonpolar content guide the identification of folded and prion proteins. Biochemistry 59, 1881\u0026ndash;1895, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsodikov, O. V., Record, M. T., Jr. \u0026amp; Sergeev, Y. V. Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem. 23, 600\u0026ndash;609, (2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2734168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2734168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, no direct information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;140\u003c/sub\u003e interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63\u0026ndash;94 residues. Longer RNCs (apoHmp\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;189\u003c/sub\u003e) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.\u003c/p\u003e","manuscriptTitle":"Nascent Chains Derived from a Foldable Protein Sequence Interact with Specific Ribosomal Surface Sites near the Exit Tunnel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-13 23:21:41","doi":"10.21203/rs.3.rs-2734168/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-07-06T09:46:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-03T15:23:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b9e9a9c6-175c-4d30-9c3b-6e1e624072df","date":"2023-07-03T07:54:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-05-04T15:23:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4f130647-81ec-41c7-9d17-34f5085cff02","date":"2023-04-23T17:16:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0380b696-e197-4400-a2b1-6d13d6861543","date":"2023-04-18T15:55:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-18T15:10:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-12T08:05:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-04-12T05:07:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-04-12T05:04:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-03-25T03:16:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34fde011-9976-40f8-9def-f99e3e940273","owner":[],"postedDate":"April 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":20633172,"name":"Biological sciences/Biophysics/Biopolymers in vivo"},{"id":20633173,"name":"Biological sciences/Cell biology/Protein folding"},{"id":20633174,"name":"Biological sciences/Biochemistry"},{"id":20633175,"name":"Biological sciences/Biophysics"},{"id":20633176,"name":"Biological sciences/Chemical biology"}],"tags":[],"updatedAt":"2024-06-01T13:02:33+00:00","versionOfRecord":{"articleIdentity":"rs-2734168","link":"https://doi.org/10.1038/s41598-024-61274-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-29 13:02:33","publishedOnDateReadable":"May 29th, 2024"},"versionCreatedAt":"2023-04-13 23:21:41","video":"","vorDoi":"10.1038/s41598-024-61274-1","vorDoiUrl":"https://doi.org/10.1038/s41598-024-61274-1","workflowStages":[]},"version":"v1","identity":"rs-2734168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2734168","identity":"rs-2734168","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}