PMID:20381501

Citation	Aponte, RA, Zimmermann, S and Reinstein, J (2010) Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity.J. Mol. Biol. 399:154-67
Abstract	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.
Links	PubMed Online version:10.1016/j.jmb.2010.03.060
Keywords	Adenosine Triphosphatases; Adenosine Triphosphate; Directed Molecular Evolution; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Hydrolysis; Models, Molecular; Mutation; Protein Conformation; Protein Folding
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Phenotype of	Taxon Information	Genotype Information (if known)	Condition Information	OMP ID	OMP Term Name	ECO ID	ECO Term Name	Notes	Status
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: NCBI Taxon ID: 83333	Genotype of Reference Strain: DnaK Genotype of Experimental Strain : Cd35	Reference Condition:					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, Table 3.
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