A Proteome-Wide Map of Chaperone-Assisted Protein Refolding in the Cytosol

Abstract

SUMMARYThe 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 proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired within an isotope-labeling strategy to globally monitor the structures of refolding E. 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 indirect measures like 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 both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations.HIGHLIGHTS-New proteomic methods are developed to probe protein refolding globally and sensitively in a complex background-The results revise the consensus model of which E. coli proteins require the GroEL chaperonin for efficient refolding-DnaK (Hsp70) and GroEL refold largely the same clientele, suggesting that these distinct chaperones share a common unifying mechanism-A small cohort of proteins cannot be fully restored by chaperones. These chaperone- nonrefolders tend to be involved in tRNA aminoacylation and glycolysis, and may have kinetically-trapped native states

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional

“…Peptides were acidified, desalted with Sep-Pak C18 1 cc Vac Cartridges, dried down, and resuspend in 0.1% formic acid, as previously described 71 . LC-MS/MS acquisition was conducted on a Thermo Ultimate3000 UHPLC system with an Acclaim Pepmap RSLC C18 column (75 μm × 25 cm, 2 μm, 100 Å) in line with a Thermo Q-Exctive HF-X Orbitrap, identically as previously described 71 .…”

“…As described in Ref. 71, one set was supplemented with 0.5 mM [ 13 C 6 ]L-Arginine and 0.4 mM [ 13 C 6 ]L-Lysine and the other with 0.5 mM L-Arginine and 0.4 mM L-Lysine. Cells were cultured at 37°C with agitation (220 rpm) to a final OD 600 of 0.8.…”

“…E. coli K12 cells (NEB) were grown in 2 sets of 3 × 50 mL (biological triplicates) of in-house prepared MOPS EZ rich media(-Arginine/-Lysine) from saturated overnight cultures with a starting OD 600 of 0.05. Similar to reported elsewhere 71 , one set was supplemented with 0.5 mM [ 13 C 6 ]L-Arginine and 0.4 mM [ 13 C 6 ]L-Lysine and the other with 0.5 mM L-Arginine and 0.4 mM L-Lysine. Cells were cultured at 37°C with agitation (220 rpm) to a final OD 600 of 0.8.…”

“…Raw normalized extracted ion intensity data for the identified peptides were exported from the .pdResult file using a three-level hierarchy (protein > peptide group > consensus feature). These data were further processed utilizing custom Python analyzer scripts (available on GitHub, and described in depth previously 49,71 ). Briefly, normalized ion counts were collected across the refolded replicates and the native replicates for each successfully identified peptide group.…”

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

“…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 …”