To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. Spy is unlike the structure of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors.

SUMMARY Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of proteins to antibiotic resistance. It allows the identification of stabilizing mutations within proteins. The large majority of mutants selected for improved antibiotic resistance are stabilized both thermodynamically and kinetically, indicating that similar principles govern stability in vivo and in vitro. The approach requires no prior structural or functional knowledge and allows selection for stability without a need to maintain function. Mutations that enhance thermodynamic stability of the protein Im7 map overwhelmingly to surface residues involved in binding to colicin E7, implying that evolutionary pressures that drive Im7-E7 complex formation may have compromised the stability of the isolated Im7 protein.

Significance For proteins, function is generally associated with order. Some proteins, however, are at least partially disordered. Because proteins tend to evolve into disorder in the absence of selection, it has been difficult to establish any significance of disorder for protein function. Here, we isolate a constitutively active variant of the normally acid-activated, conditionally disordered chaperone HdeA. We find this mutant to be largely destabilized, partially unstructured, and monomeric at a concentration at which it prevents the aggregation of a client protein. Our data therefore provide experimental evidence for the significance of partial disorder in protein function.