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The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain’s intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis–mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.
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This content will become publicly available on March 14, 2026
Protein misfolding involving entanglements provides a structural explanation for the origin of stretched-exponential refolding kinetics
Stretched-exponential protein refolding kinetics, first observed decades ago, were attributed to a nonnative ensemble of structures with parallel, non-interconverting folding pathways. However, the structural origin of the large energy barriers preventing interconversion between these folding pathways is unknown. Here, we combine simulations with limited proteolysis (LiP) and cross-linking (XL) mass spectrometry (MS) to study the protein phosphoglycerate kinase (PGK). Simulations recapitulate its stretched-exponential folding kinetics and reveal that misfolded states involving changes of entanglement underlie this behavior: either formation of a nonnative, noncovalent lasso entanglement or failure to form a native entanglement. These misfolded states act as kinetic traps, requiring extensive unfolding to escape, which results in a distribution of free energy barriers and pathway partitioning. Using LiP-MS and XL-MS, we propose heterogeneous structural ensembles consistent with these data that represent the potential long-lived misfolded states PGK populates. This structural and energetic heterogeneity creates a hierarchy of refolding timescales, explaining stretched-exponential kinetics.
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- Award ID(s):
- 2031584
- PAR ID:
- 10616337
- Publisher / Repository:
- AAAS
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 11
- Issue:
- 11
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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