Genetic selection designed to stabilize proteins uncovers a chaperone called Spy

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Abstract
Genetic selection uncovers a novel periplasmic chaperone called Spy, whose expression is strongly induced by conditions that cause protein unfolding. Spy can suppress protein aggregation and promote refolding in an ATP-independent manner in vitro. The crystal structure of Spy reveals a thin, cradle-shaped dimer that might shield aggregation-prone regions exposed in unfolded substrates. 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. The structure of Spy is unlike that 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.

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