PT  - JOURNAL ARTICLE
AU  - Thomas E. Wales
AU  - Aleksandra Pajak
AU  - Alžběta Roeselová
AU  - Santosh Shivakumaraswamy
AU  - Steven Howell
AU  - F. Ulrich Hartl
AU  - John R. Engen
AU  - David Balchin
TI  - Resolving chaperone-assisted protein folding on the ribosome at the peptide level
AID  - 10.1101/2022.09.23.509153
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.09.23.509153
4099  - http://biorxiv.org/content/early/2022/09/23/2022.09.23.509153.short
4100  - http://biorxiv.org/content/early/2022/09/23/2022.09.23.509153.full
AB  - The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. We used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. On the ribosome, the nascent polypeptide folds 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. We find that partially-folded nascent chains recruit the chaperone Trigger factor, which uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. In addition, we comprehensively mapped dynamic 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 de novo protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.Competing Interest StatementThe authors have declared no competing interest.