Systematic Interrogation of Protein Refolding Under Cellular-Like Conditions

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