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.

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The aldol reaction is recognized as an archetypal method to form carbon‐carbon bonds. Base‐catalyzed aldol reactions of aryl aldehydes with aryl methyl ketones typically produce condensation products (chalcones). However, 2‐pyridinecarboxaldehye, 2‐quinolinecarboxaldehyde, and 2‐ and 3‐fluorobenzaldehyde undergo tandem aldol‐Michael reactions with aryl enolates to yield conjugate addition products. To elucidate the different reactivities of aldol products formed in the reactions of benzaldehyde and 2‐pyridinecarboxaldehyde with the sodium enolate of acetophenone, density functional theory (DFT) calculations were performed to compare the energies of conformational isomers of the intermediates formed in the reaction, the energies of the corresponding condensation products, and the kinetic barriers to dehydration and conjugate addition. Computational results support the formation of the trans isomer of the condensation product in base‐catalyzed aldol reactions of either benzaldehyde or 2‐pyridinecarboxaldehyde with acetophenone. The susceptibility of the condensation product to conjugate addition is determined to result from the lower LUMO energy of trans‐1‐phenyl‐3‐(2‐pyridinyl)‐2‐propen‐1‐one. Consequently, the kinetic barrier for conjugate addition of a second equivalent of enolate with the condensation product of 2‐pyridinecarboxaldehyde is found to be significantly lower. Similar results were obtained for the reactions of fluorobenzaldehydes with aryl enolates.

Decades of research on protein folding have primarily focused on a privileged subset of small proteins that can reversibly refold out of a denaturant. However, these studies are not representative of the complexity of natural proteomes, which consist of many proteins with more sophisticated architectures. Here, we introduce an experimental approach to probe protein refolding for whole proteomes. We accomplish this by first unfolding and refolding E. coli lysates, and then interrogating the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions. Using mass spectrometry, we globally analyze the digestion patterns to assess structural differences between native and “refolded” proteins. These studies show that roughly half of the E. coli proteome cannot reassemble their native states following chemical denaturation. Our results imply that thermodynamics alone cannot specify the native structures of all proteins, signaling a pervasive role for kinetic control in shaping protein biogenesis.One Sentence SummaryMany proteins cannot put themselves back together again after being taken apart, suggesting that their sequences do not encode sufficient information to fold them.