Non-Refoldability is Pervasive Across the E. coli Proteome

Decades of protein folding research have focused on a small, privileged subset of proteins that can reversibly refold upon denaturation. However, these proteins are not necessarily representative of the complexity of natural proteomes, which consist of many proteins with more sophisticated architectures. Here, we introduce an experimental approach to probe the refolding of whole proteomes. To accomplish this, we first unfold and refold E. coli lysates, and then interrogate the resulting protein structures usin a permissive enzyme that preferentially cleaves at more flexible regions. Using mass spectrometry, we analyze the digestion patterns to globally assess structural differences between native and “refolded” proteins. These studies reveal that many soluble proteins are incapable of navigating back to their native structures upon chemical denaturation, highlighting the role of exogenous factors and processes such as molecular chaperones or co-translational folding for efficient protein folding.