In vitro evolution of uracil glycosylase towards DnaKJ and GroEL binding evolves different misfolded states

Abstract

AbstractNatural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time scale and is driven by specific outcomes, such as binding to a high-affinity bait. Here, we used directed in vitro evolution to determine how DnaKJ and GroEL change the sequence and structure space of the E. coli protein Uracil glycosylase (eUNG). Using yeast surface display, we generated a random library of eUNG and selected it for binding to chaperones GroEL or DnaK+DnaJ+ATP (DnaKJ). We found that these chaperones select and enrich for mutations causing eUNG to misfold. Evolution in the presence of either chaperone thus resulted in the accumulation of mutations in buried and conserved positions, with a tendency to increase the positive charge. However, GroEL was selected for more exposed and less structured proteins that were highly sensitive to protease cleavage, while DnaKJ was selected for partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. In a more general context, our results show that ATP-fueled chaperones can purge promiscuous misfolded protein mutants from the system, thereby avoiding their potentially detrimental effects, such as forming wrong interactions with native proteins or form aggregates, compromising cellular function.