Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat Struct Mol Biol 18

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. The folding of many proteins is assisted by molecular chaperones and other folding helpers in the cell1. Many chaperones act by inhibiting off-pathway events, such as aggregation, and serve a broad range of substrates in a stoichiometric manner. In vitro assays for chaperone activity are thus almost by necessity relatively insensitive, making these assays more usefu