Nonrefoldability is Pervasive Across the E. coli Proteome

Abstract

Decades of research on protein folding have primarily focused on a subset of small proteins that can reversibly refold from a denatured state. However, these studies have generally not been representative of the complexity of natural proteomes, which consist of many proteins with complex architectures and domain organizations. Here, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes using mass spectrometry-based proteomics. Our study covers the majority of the soluble E. coli proteome expressed during log-phase growth, and among this group, we find that one-third of the E. coli proteome is not intrinsically refoldable on physiological time scales, a cohort that is enriched with certain fold-types, domain organizations, and other biophysical features. We also identify several properties and fold-types that are correlated with slow refolding on the minute time scale. Hence, these results illuminate when exogenous factors and processes, such as chaperones or cotranslational folding, might be required for efficient protein folding.

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

“…In preliminary experiments, we tested whether cyto-serum would be suitable for global refolding experiments by measuring the levels of aggregation that accrue after 2 h. Pelleting assays detected low but non-zero levels of aggregation (6 ± 2% of protein), in contrast with the buffer conditions we had previously devised (20 mM Tris pH 8.2, 100 mM NaCl, 2 mM MgCl2, 1 mM DTT) that result in very low levels of precipitation (3 ± 1%, Figure 1-figure supplement 2D). This 3% increase in aggregation is close to what we previously observed for refolding in a defined buffer at neutral pH (To et al, 2021;Wang et al, 2017), thereby confirming that alkaline pH helps suppress aggregation, and that the cytosolic components do not increase aggregation levels beyond an expected effect from pH. To further investigate aggregation (including smaller soluble non-precipitating aggregation), we performed sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry (MP) on these refolding reactions (Figure 1-figure supplement 3).…”

“…(2) our previous study (To et al, 2021) confirmed that refolded/native protein abundance ratios were equal to unity at a frequency higher than the false discovery rate.…”

“…A representation of all samples prepared for this study is presented in Figure 1–figure supplement 1. We note here that LiP-MS studies typically prepare a series of parallel ‘control’ samples in which PK is withheld; these samples are then used for standard quantitative proteomics experiments to measure protein abundance differences across conditions (Feng et al, 2014; To et al, 2021). We opted to not perform this for the current study for the following reasons: (1) there is no practical way native and refolded samples that are compared to each other can have different protein abundances given that they are derived from the same lysates; indeed, the samples compared to each other for these studies are compositionally identical and differ only in history; (2) our previous study (To et al, 2021) confirmed that refolded/native protein abundance ratios were equal to unity at a frequency higher than the false discovery rate.…”

“…Indeed, many proteins that were presumed to be obligate chaperonin clients based on their enrichment on chaperonin co-precipitation studies were found to remain soluble in vivo during GroE knock-down (Fujiwara et al, 2010). Furthermore, a recent study (To et al, 2021) estimated that a third of soluble E. coli proteins are intrinsically nonrefoldable, meaning they cannot fully reassume their native forms following complete denaturation, even under conditions without appreciable precipitation. However, how many (and what kinds) of intrinsically nonrefoldable proteins can be rescued by chaperones -as opposed to requiring cotranslational folding (Fedorov and Baldwin 1997;Frydman et al, 1999;Liu et al, 2019) -is not known.…”

Is Protein Folding a Thermodynamically Unfavorable, Active, Energy-Dependent Process?

“…To our knowledge, no representative samples of proteins have been studied under this angle until very recently. However, in a recent proteome-wide study, protease-resistance assay was used in combination with quantitative mass-spectrometry to show that about 50% of the proteins in E. coli cell lysates could not refold into their native states following chemical denaturation, even when the conditions were optimized for refolding [ 71 ]. These findings indicate that non-refoldability in vitro is a general characteristic of at least this bacterial proteome, especially, taking into consideration that the completeness of unfolding was not monitored in these experiments.…”