Protein folding begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. Cotranslational folding and chaperone activity are critical for efficient protein biogenesis, but how they direct the maturation pathway of nascent proteins remains poorly understood. I will present our results on the cotranslational, chaperone-assisted folding pathway of <i>Escherichia coli</i> dihydrofolate reductase (DHFR), defined at the peptide level by hydrogen/deuterium exchange (HDX)-mass spectrometry. By HDX analysis of a series of stalled ribosome:nascent chain complexes, we reconstructed the detailed cotranslational folding pathway of DHFR. Our novel approach resolves subtle local conformational differences between ribosome-bound folding intermediates, while simultaneously reporting on the dynamic behaviour and interactions of ribosomal proteins and bound chaperones. On the ribosome, the nascent polypeptide folds along an unanticipated pathway, via structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing C-terminal sequences remain confined in the ribosome exit tunnel, suggesting a general role for the tunnel in facilitating folding. Molecular chaperones are a ubiquitous feature of the cellular protein folding landscape, and we find that partially-folded nascent chains recruit the endogenous chaperone Trigger factor. We show that Trigger factor uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. As a result, the nascent chain bound by Trigger factor is poised to complete folding immediately upon emergence of the C-terminus from the exit tunnel. We further mined our HDX MS data to study ribosome dynamics at the peptide level. We comprehensively map interactions between the nascent chain and ribosomal proteins, tracing the path of the emerging polypeptide during synthesis. Our work provides a high-resolution description of <i>de novo</i> protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.

Resolving chaperone-assisted protein folding on the ribosome at the peptide level

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