A proteome-wide map of chaperone-assisted protein refolding in a cytosol-like milieu

Abstract

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.

DomainMapper: Accurate domain structure annotation including those with non‐contiguous topologies

“…For these categories, non‐contiguity is enriched 6.9‐fold, 5.6‐fold, and 5‐fold, respectively. The high propensity of non‐contiguity in these enzymes is consistent with their general inability to reversibly refold, 14,15 and is consistent with the conjecture that they co‐evolved with the emergence of protein synthesis by translation 32 …”

“…We initially became intrigued with NC domains because recent studies found that E. coli proteins with NC domain topologies are generally less refoldable and rely more heavily on chaperones to assist their assembly. 14,15 These findings are consistent with the general understanding that long-range contacts in proteins are more challenging and slower to form. 16 With the recent upgrade to v2.0, 17 every protein record in Uniprot possesses a Family & Domains section which draws data from InterPro, 18 an EMBL-EBI database that compiles 13 protein signature databases including CATH-Gene3D, SCOP, and Pfam, among others.…”

“…For these categories, non-contiguity is enriched 6.9-fold, 5.6-fold, and 5-fold, respectively. The high propensity of non-contiguity in these enzymes is consistent with their general inability to reversibly refold, 14,15 and is consistent with the conjecture that they co-evolved with the emergence of protein synthesis by translation. 32 Several X-groups associated with integral membrane transporters are quite enriched for non-contiguity (Figure 5b), 33 including neurotransmitter-gated ionchannel folds (17-fold), proton glutamate symport folds (13-fold), and voltage-gated ion channels (8-fold).…”

“…Though it is simple to code a basic script for this task, conventional parsing strategies will exclude or mis‐annotate domains with non‐contiguous (NC) residue ranges, especially when there are insertional (IS) domains residing within them. We initially became intrigued with NC domains because recent studies found that E. coli proteins with NC domain topologies are generally less refoldable and rely more heavily on chaperones to assist their assembly 14,15 . These findings are consistent with the general understanding that long‐range contacts in proteins are more challenging and slower to form 16 …”

A Newly Identified Class of Protein Misfolding in All-atom Folding Simulations Consistent with Limited Proteolysis Mass Spectrometry

“…To experimentally test for the existence of these misfolded states, we unfolded purified 4-Diphosphocytidyl-2-C-Methyl-D-Erythritol Kinase (ispE) in 6 M guanidinium chloride, initiated refolding by dilution, and then probed the protein’s conformation with Limited Proteolysis Mass Spectrometry 18,19 (LiP-MS, see SI Methods). LiP-MS allows us to pinpoint subtle structural differences between folded and misfolded conformations 2,20 . Based on the predicted timescales from simulations, we allowed ispE three relatively long refolding times (1 h, 10 h, and 24 h, Table S8 and Supplementary Data) before probing it with limited proteolysis.…”