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

ABSTRACT: 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.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: John R. Engen

LAB HEAD: John R. Engen

PROVIDER: PXD036945 | Pride | 2024-07-12

REPOSITORIES: Pride

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			Action	DRS
	01_FL_DHFR_1000s1.raw.zip	Raw
	01_FL_DHFR_1000s2.raw.zip	Raw
	01_FL_DHFR_1000s3.raw.zip	Raw
	01_FL_DHFR_100s1.raw.zip	Raw
	01_FL_DHFR_100s2.raw.zip	Raw

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

Wales Thomas E TE Pajak Aleksandra A Roeselová Alžběta A Shivakumaraswamy Santosh S Howell Steven S Kjær Svend S Hartl F Ulrich FU Engen John R JR Balchin David D

Nature structural & molecular biology 20240710

Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen-deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unan ...[more]

PMID: 38987455

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