Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity.

We improved the DnaK molecular chaperone system for increased folding efficiency towards two target proteins, by using a multi-parameter screening procedure. First, we used a folding-deficient C-terminal truncated chloramphenicol acetyl transferase (CAT_Cd9) to obtain tunable selective pressure for enhanced DnaK chaperon function in vivo. Second, we screened selected clones in vitro for CAT_Cd9 activity after growth under selective pressure. We then analyzed how these variants performed as compared to wild type DnaK towards folding assistance of a second target protein; namely, chemically denatured firefly luciferase. A total of 11 single point DnaK mutants and 1 truncated variant were identified using CAT_Cd9 as the protein target, while 4 of the 12 selected variants showed improved luciferase refolding in vitro. This shows that improving the DnaK chaperone by using a certain target substrate protein, does not necessarily result in a loss or reduction in its ability to assist other proteins. Of the 12 identified mutations, half were clustered in the nucleotide binding domain, and half in the lid domain (LD) of DnaK. The truncated variant is characterized by a 35-residue C-terminal truncation (Cd35) and exhibited the highest improvement for luciferase refolding. Cd35 showed a 7-fold increase in initial refolding rate for denatured luciferase and resulted in a 5-fold increase in maximal luminescence as compared to wild type DnaK. Given that the best in vitro performing mutants contained LD substitutions, and that the LD is not involved in ATP binding, ATP hydrolysis or client protein association, but is involved in allosteric regulation of the chaperone cycle, we propose that improved DnaK variants result in changes to allosteric domain communication, ultimately retuning the ATP-dependent chaperone cycle.