A 2024 hydrogen–deuterium exchange mass spectrometry (HDX-MS) study defined the first peptide-resolution cotranslational folding pathway of a chaperone-assisted substrate, Escherichia coli dihydrofolate reductase (DHFR), showing that the ribosome enables otherwise unstable folding intermediates by confining destabilizing C-terminal sequences inside the exit tunnel, and that the chaperone trigger factor (TF) binds partially folded states without disrupting them. The open problem is to turn this single-substrate picture into general mechanistic rules: which sequence and topology classes exploit exit-tunnel occlusion to stabilize intermediates; what quantitative rules govern TF recruitment to diverse nascent chains in the cell; what the TF peptidyl-prolyl isomerase domain (PPD) contributes to de novo folding; and what determines whether proximity to the ribosome stabilizes or destabilizes local structure and how this affects cofactor loading and domain assembly in multidomain proteins. Recent work (2024–2026) has delivered major partial progress on each front, but no cross-topology rule, no in-vivo recruitment rules, no PPD mechanism, and no determinants-level rule for whether ribosome proximity stabilizes versus destabilizes nascent structure exist, so the core remains open.

Background

Proteins begin to fold while they are being synthesized: the nascent polypeptide chain (NC) emerges from the ribosome through a roughly 10-nanometer exit tunnel and can form structure on the ribosome surface before synthesis is complete. This is called cotranslational folding, and it differs from the refolding of a full-length denatured chain, which is what most classical in-vitro protein-folding studies measured. Studying it requires ribosome–nascent-chain complexes (RNCs): ribosomes stalled at a chosen peptide length with the NC attached, which can be purified and probed as snapshots at defined elongation stages. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) measures how fast backbone amide hydrogens in each peptide segment exchange with deuterated solvent: segments folded or protected exchange slowly, flexible or exposed segments exchange fast, yielding a local (peptide-level) readout of structure and dynamics for all proteins in a complex at once, including both the NC and ribosomal proteins. In bacteria, the first chaperone to meet the nascent chain is trigger factor (TF), a three-domain protein that docks near the tunnel exit: a ribosome-binding domain (RBD) attaches to the ribosomal protein L23 at the tunnel mouth, a substrate-binding domain (SBD) holds client polypeptides, and a peptidyl-prolyl cis/trans isomerase domain (PPD) catalyzes the isomerization of proline peptide bonds, an activity that genetic experiments showed is not essential for folding of cytosolic proteins in E. coli (Kramer et al., 2004), leaving the PPD's actual role in de novo folding unexplained ('enigmatic'). Before 2024, high-resolution views of cotranslational folding came mainly from NMR of small domains and from force spectroscopy, and the field had established two seemingly opposing effects of the ribosome: proximity to the negatively charged ribosome surface can thermodynamically destabilize some folded nascent domains, yet ribosome tethering can stabilize folding intermediates relative to the native state (as shown for an immunoglobulin-like domain). The 2024 study by Wales, Pajak, Roeselová and colleagues supplied the missing peptide-level view for one substrate, E. coli DHFR (a model enzyme with a discontinuous domain, whose central beta-sheet is built from sequence segments far apart in the chain, one contributed by the C-terminal region). It found that DHFR folds cotranslationally through intermediates not populated during refolding from denaturant; that the ribosome enables these intermediates because the still-untranslated, conformationally destabilizing C-terminal sequence stays confined inside the exit tunnel, buffering the nascent chain against the entropic destabilization that disordered termini cause; that TF engages these fragile intermediates through a chemically heterogeneous, dynamic interface without perturbing them; and that NC contacts with ribosomal proteins (L29 at the exit port, L23 further out) trace the emerging chain's path. The authors proposed that occlusion of discontinuous C-terminal segments 'may be a generic mechanism' and that ribosome interactions may positively modulate N-terminal domain function in multidomain proteins, for example by promoting cofactor loading during synthesis - proposals, not demonstrated generalities. Sibling work from the same laboratory (Roeselová et al., Molecular Cell 2024) reconstituted the bacterial chaperone cascade on RNCs, showing that TF recognizes compact intermediates exposing an extensive unfolded surface and gates DnaJ/DnaK access, with none of these chaperones destabilizing cotranslational intermediates; a companion cryo-EM/HDX-MS study of GroEL/ES showed the chaperonin can partially encapsulate ribosome-tethered domains, with TF and GroEL co-accommodated on long nascent chains; and single-particle tracking in living E. coli has measured TF's in-vivo binding modes on actively translating ribosomes (roughly 1-second and 50-millisecond residence components, with competition against the signal recognition particle), giving the first in-vivo residence-time data but not sequence- or topology-resolved recruitment rules (Hävermark et al., PNAS 2025).

Problem Statement

Establish general mechanistic rules for how the ribosome and trigger factor jointly determine the outcome of de novo (cotranslational) protein folding, at peptide resolution, generalizing the single-substrate DHFR pathway of Wales et al. (2024). Concretely, the source and subsequent literature highlight four linked open directions toward this single objective: (1) Generality of ribosome-enabled intermediate stabilization - determine which classes of proteins exploit the ribosome to stabilize folding intermediates – whether by confinement of destabilizing (e.g., discontinuous or C-terminal) sequences in the exit tunnel, as for DHFR, or by ribosome tethering and surface electrostatics, as shown for an immunoglobulin-like domain – and which do not, as a function of topology and domain architecture. (2) TF recruitment rules - establish quantitative rules for TF engagement on the ribosome: how the balance of hydrophobic and electrostatic interactions, competition for the L23 docking region (including with basic nascent sequences), ionic strength, and the conformational state of the nascent chain jointly control TF binding kinetics and residence times across structurally diverse nascent chains, under physiological (in-vivo) conditions. (3) PPD function - determine the specific mechanistic contribution of TF's peptidyl-prolyl isomerase domain to cotranslational folding, given that its catalytic activity is genetically dispensable. (4) Stabilize-versus-destabilize determinants - identify the protein- and sequence-specific determinants that dictate whether proximity to the ribosome (and to TF) stabilizes or destabilizes local nascent structure, and quantify how this modulation affects downstream maturation such as cofactor loading and domain docking/assembly in multidomain proteins. These four directions are linked sub-questions that jointly constitute the single objective above, and each is independently refutable: none alone resolves the objective, and partial coverage of the four is reportable progress but not a resolution. The objective is met only once every direction yields a definite, falsifiable claim – a rule, mechanism, or determinant set that makes specific predictions and could be overturned by observation – rather than an open survey; a refutation inside one direction settles that direction and leaves the others open.

References

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