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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 September 24, 2026
DnaK refolds denatured proteins by actively pulling out their misfolded structural elements
a) Abstract DnaK is a prokaryotic Hsp70 chaperone, with numerous functions in helping to fold nascent polypeptides and more generally in proteostasis. It also restores native structures to heat-shocked proteins in an ATP-hydrolysis-dependent manner. The structures of DnaK complexes with nucleotides, co-chaperones andsmallpeptides have already been resolved. However, there are no structures of DnaK complexes with larger, mostly folded substrates, such as firefly luciferase (Fluc, 61 kDa), which impedes the understanding of the mechanism through which DnaK refolds such large proteins. Here, we generated a model of a DnaK-firefly luciferase complex with Alphafold3, and examined its dynamics with all-atom molecular dynamics simulations. In this complex, Fluc is immobilized under the DnaK alpha-helical lid against the NBD, not the SBDβ, contrary to the data reported in the literature for model peptides. The DnaK lid is positioned strategically over Fluc’s helix 405-411, which we recently determined to be the first (and likely the only) helix melted in Fluc at 42 °C. We simulated the interaction between DnaK and the helix in its native and misfolded state and found that during the lid translocation toward the SBDβ, only the melted helix follows the lid and is actively pulled out from Fluc, while the native helix is not dislocated. These observations suggest a new model for the DnaK chaperone mechanism, where the alpha helical lid forms hydrogen bonds to the protein segment to be structurally tested. Lid pulls out only highly deformable misfolded helices, allowing them to refold into their native structures, and does not pull out those that are correctly folded because they are not deformable. Broader Audience Statementc) DnaK is a model chaperone, which can reactivate thermally denatured proteins. Even though a plethora of significant findings about DnaK structure, dynamics and interactions with its co-chaperone have been accumulated over 30 years, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited the ability of the Alphafold3 platform to generate an atomistic model for a complex between DnaK and Firefly luciferase and used molecular dynamics simulations to directly capture how DnaK may assist denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism.
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- Award ID(s):
- 2118357
- PAR ID:
- 10649887
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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