The demonstration by Nilsson and coworkers (Cell Reports, 2015) that the small zinc-finger domain ADR1a can fold deep inside the vestibule of the ribosome exit tunnel posed two forward questions: (i) a quantitative mapping between the arrest-peptide full-length fraction readout fFLf_{FL} and the mechanical folding force in piconewtons, and (ii) general rules by which a protein domain's size and shape dictate where (inside the tunnel, at its vestibule, or outside) it folds during translation — a question to which later work has added candidate determinants such as the domain's native stability, translation rate, and tunnel-region energetics, without establishing the rules themselves. Later work has substantially answered (i): optical-tweezer force-release measurements and a Bell-model analysis of arrest-release kinetics now connect fFLf_{FL} to forces of order 10 pN. What remains open is (ii): no validated general rule set exists that predicts, from a domain's size and shape, where it folds in relation to the exit tunnel. This problem asks for such rules.

Background

Proteins are synthesized by the ribosome, a two-subunit RNA–protein machine whose large subunit contains the exit tunnel, a roughly 10-nanometer-long, mostly water-filled channel with a narrow upper part and a wider lower part called the vestibule (the last ~3 nm, wide enough for tertiary folding). The growing nascent polypeptide traverses this tunnel before emerging into the cytosol, so the first folding events of every protein happen while it is still attached to the ribosome — cotranslational folding. A key experimental tool is the arrest-peptide force assay: a stalling sequence such as the E. coli SecM arrest peptide halts translation; if a domain folding on the outside end of the nascent chain pulls on the peptide, the stall is partially relieved. The readout is fFLf_{FL}, the fraction of full-length product relative to arrested product, measured as a function of the tether length LL between the tunnel exit and the folding domain (force profile analysis, FPA). Nilsson et al. (2015) combined FPA, single-ribosome FRET (fluorescence resonance energy transfer, a distance-sensitive spectroscopy), coarse-grained molecular dynamics, and cryo-electron microscopy of ribosome–nascent-chain complexes to show that the 29-residue zinc-finger domain ADR1a undergoes its cotranslational folding transition while buried deep in the vestibule — the first demonstration that a compact domain can fold inside the ribosome exit tunnel, which until then had been seen only to support alpha-helical secondary structure and partial compaction. Larger model domains, such as Top7, fold outside the tunnel; membrane-protein helices and translocated charged segments pull on arrest peptides as well (Ismail et al. 2012; Cymer and von Heijne 2013). The paper closed by asking how the size and shape of a domain dictate where it folds relative to the tunnel, and how fFLf_{FL} relates quantitatively to force. Subsequent work established the force calibration: Goldman et al. (2015) used optical tweezers (laser-trap single-molecule force spectroscopy) to show that mechanical force alone releases SecM arrest and formulated a kinetic model of force-dependent release, and Kemp et al. (2020) directly mapped fFLf_{FL} to force by combining arrest-release kinetics with a Bell model (a standard model in which a bond's rupture rate grows exponentially with applied force), estimating a maximal cotranslational folding force of ~15 pN for the spectrin R16 domain. On folding location, later studies give building blocks of a rule set but not the rule set itself: deleting tunnel-wall protein loops (uL23, uL24) shifts where domains of a given size fold, supporting a first-order geometric 'sufficient space' criterion (Kudva et al. 2018); single-molecule experiments on ADR1a show the tunnel can accelerate and stabilize folding through electrostatic interactions rather than pure confinement (Wruck et al. 2021); all-atom simulations show the hydrophobic effect — the main driving force of folding — is about twofold weaker in the vestibule because of ordered water (Vu et al. 2021); coarse-grained simulations indicate that domain stability, topology (the connectivity and contact pattern of the folded state), and translation speed jointly determine force generation and that arrested-ribosome measurements can misrepresent in-vivo behavior (Leininger et al. 2019); rational engineering of tunnel loops tunes the folding energetics of a filamin domain (Ahn et al. 2022); and high-throughput in-vivo pathway mapping by Arrest Peptide Profiling has been demonstrated for one GTPase fold family (Chen et al. 2025). Atomistic prediction plus experimental validation of one vectorial folding pathway through non-native intermediates was achieved in 2025, with the authors noting that predicting cotranslational folding pathways in general remains challenging (Wang et al. 2025), and the human ribosome was shown to delay inter-domain docking relative to bacteria, so any rules also depend on the ribosome itself (Pellowe et al. 2025). Reviews in 2024 still list the determinants of folding location among the open 'white spots' of the field (Kolář et al. 2024; Samatova et al. 2024).

Problem Statement

Determine general, quantitative rules by which a protein domain's size and folded-state shape dictate where along the ribosome exit tunnel (upper tunnel, vestibule, exit port, or fully outside) the domain undergoes its cotranslational folding transition. The dependence of interest is on the domain's size and folded-state shape; the further determinants noted in the Background — native stability, translation rate, tunnel-region energetics, and ribosome identity — are context. The rules must cover both small domains that can fold inside the tunnel (as ADR1a does) and larger domains that fold outside it. The companion calibration question posed alongside this problem in 2015 — mapping the arrest-peptide readout fFLf_{FL} quantitatively onto forces in piconewtons — has been answered by optical-tweezer force-release measurements and Bell-model kinetic analyses and is not part of what remains to be solved.

References

  1. Nilsson OB, Hedman R, Marino J, Wickles S, Bischoff L, Johansson M, Müller-Lucks A, Trovato F, Puglisi JD, O'Brien EP, Beckmann R, von Heijne G. Cotranslational Protein Folding inside the Ribosome Exit Tunnel. Cell Reports 2015;12(10):1533-1540. DOI: 10.1016/j.celrep.2015.07.065. https://doi.org/10.1016/j.celrep.2015.07.065
  2. Ismail N, Hedman R, Schiller N, von Heijne G. A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Nature Structural & Molecular Biology 2012;19:1018-1022. DOI: 10.1038/nsmb.2376. https://doi.org/10.1038/nsmb.2376
  3. Cymer F, von Heijne G. Cotranslational folding of membrane proteins probed by arrest-peptide-mediated force measurements. Proceedings of the National Academy of Sciences 2013;110:14640-14645. DOI: 10.1073/pnas.1306787110. https://doi.org/10.1073/pnas.1306787110
  4. Goldman DH, Kaiser CM, Milin A, Righini M, Tinoco I, Bustamante C. Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo. Science 2015;348(6233):457-460. DOI: 10.1126/science.1261909. https://doi.org/10.1126/science.1261909
  5. Kemp G, Kudva R, de la Rosa A, von Heijne G. Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays. Journal of Molecular Biology 2019;431:1308-1314. DOI: 10.1016/j.jmb.2019.01.043. https://doi.org/10.1016/j.jmb.2019.01.043
  6. Leininger SE, Trovato F, Nissley DA, O'Brien EP. Domain topology, stability, and translation speed determine mechanical force generation on the ribosome. Proceedings of the National Academy of Sciences 2019;116:5523-5532. DOI: 10.1073/pnas.1813003116. https://doi.org/10.1073/pnas.1813003116
  7. Kudva R, Tian P, Pardo-Avila F, Carroni M, Best RB, Bernstein HD, von Heijne G. The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding. eLife 2018;7:e36326. DOI: 10.7554/eLife.36326. https://doi.org/10.7554/eLife.36326
  8. Kemp G, Nilsson OB, Tian P, Best RB, von Heijne G. Cotranslational folding cooperativity of contiguous domains of α-spectrin. Proceedings of the National Academy of Sciences 2020;117:14119-14126. DOI: 10.1073/pnas.1909683117. https://doi.org/10.1073/pnas.1909683117
  9. Wruck F, Tian P, Kudva R, Best RB, von Heijne G, Tans SJ, Katranidis A. The ribosome modulates folding inside the ribosomal exit tunnel. Communications Biology 2021;4:511. DOI: 10.1038/s42003-021-02055-8. https://doi.org/10.1038/s42003-021-02055-8
  10. Vu QV, Jiang Y, Li MS, O'Brien EP. The driving force for co-translational protein folding is weaker in the ribosome vestibule due to greater water ordering. Chemical Science 2021;12:11851-11857. DOI: 10.1039/d1sc01008e. https://doi.org/10.1039/d1sc01008e
  11. Ahn M, Włodarski T, Mitropoulou A, Chan SHS, Sidhu H, Plessa E, Becker TA, Budisa N, Waudby CA, Beckmann R, Cassaignau AME, Cabrita LD, Christodoulou J. Modulating co-translational protein folding by rational design and ribosome engineering. Nature Communications 2022;13:396. DOI: 10.1038/s41467-022-31906-z. https://doi.org/10.1038/s41467-022-31906-z
  12. Kolář MH, McGrath H, Nepomuceno FC, Černeková M. Three Stages of Nascent Protein Translocation Through the Ribosome Exit Tunnel. WIREs RNA 2024;15:e1873. DOI: 10.1002/wrna.1873. https://doi.org/10.1002/wrna.1873
  13. Samatova E, Komar AA, Rodnina MV. How the ribosome shapes cotranslational protein folding. Current Opinion in Structural Biology 2024;84:102740. DOI: 10.1016/j.sbi.2023.102740. https://doi.org/10.1016/j.sbi.2023.102740
  14. Gersteuer F, Morici M, Gabrielli S, Fujiwara K, Safdari HA, Paternoga H, Bock LV, Chiba S, Wilson DN. The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome. Nature Communications 2024;15:3403. DOI: 10.1038/s41467-024-46762-2. https://doi.org/10.1038/s41467-024-46762-2
  15. Chen X, Hilser VJ, Kaiser CM. Arrest Peptide Profiling resolves co-translational folding pathways and chaperone interactions in vivo. Nature Communications 2025;16:4164. DOI: 10.1038/s41467-025-61398-6. https://doi.org/10.1038/s41467-025-61398-6
  16. Wang S, Bitran A, Samatova E, Shakhnovich EI, Rodnina MV. Cotranslational protein folding through non-native structural intermediates. Science Advances 2025;11:eady2211. DOI: 10.1126/sciadv.ady2211. https://doi.org/10.1126/sciadv.ady2211
  17. Pellowe GA, Voisin TB, Karpauskaite L, Maslen SL, Roeselová A, Skehel JM, Roustan C, George R, Nans A, Kjær S, Taylor IA, Balchin D. The human ribosome modulates multidomain protein biogenesis by delaying cotranslational domain docking. Nature Structural & Molecular Biology 2025;32:2296-2307. DOI: 10.1038/s41594-025-01676-5. https://doi.org/10.1038/s41594-025-01676-5