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Analysis of stalled ribosome-nascent chain complexes by quantitative proteomics

ABSTRACT: The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. To understand how the cellular environment modulates folding, we studied the cotranslational chaperone-assisted folding of Escherichia coli dihydrofolate reductase (DHFR). To sample folding intermediates along the pathway of vectorial synthesis, we prepared a series of stalled ribosome:nascent chain complexes (RNCs) representing snapshots of DHFR folding in vivo. Proteomic analysis of RNCs revealed their composition and allowed us to define chaperone interactions as a function of NC length. These data set the stage for further studies aimed at resolving the conformation of the nascent polypeptide on the ribosome.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Steven Howell

LAB HEAD: David Balchin

PROVIDER: PXD036784 | Pride | 2025-05-06

REPOSITORIES: Pride

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			Action	DRS
	HS_1_106RNC.raw	Raw
	HS_1_106RNCdeltaTF_1.raw	Raw
	HS_1_106RNCdeltaTF_2.raw	Raw
	HS_1_106RNCdeltaTF_3.raw	Raw
	HS_1_126RNC.raw	Raw

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Publications

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 12

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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Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

Project description:bRNC Proteomics - Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

2025-08-11 | PXD055060 | Pride

Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

Project description:hRNC Proteomics - Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

2025-08-11 | PXD055069 | Pride

STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins

Project description:Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2’s β-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

2024-08-28 | PXD042896 | Pride

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

2024-07-03 | PXD048642 | Pride

Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

Project description:bRNC HDX-MS: Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

2025-08-11 | PXD055476 | Pride

Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

Project description:hRNC HDX-MS: Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

2025-08-11 | PXD055280 | Pride

Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

Project description:hRNC XL-MS: Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

2025-08-11 | PXD055251 | Pride

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

2024-07-03 | PXD048638 | Pride

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

Project description: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.

2024-07-12 | PXD036945 | Pride

A molecular network of conserved factors keeps ribosomes dormant in the egg

Project description:Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally inactive. How translation is activated during embryogenesis is poorly understood. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we find that ribosomes transition from a dormant to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four factors that form two modules: a Habp4-eEF2 module that stabilizes ribosome levels and a Dap1b/Dapl1-eIF-5a module that represses translation. Dap/Dap1b is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Thus, a developmentally programmed, unique ribosome state plays a key role in ribosome storage and translational repression in the egg.

2022-11-03 | PXD026866 | Pride

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