The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein.

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

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

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