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          Institute: MPI für medizinische Forschung     Collection: Abteilung Biomolekulare Mechanismen     Display Documents

ID: 545995.0, MPI für medizinische Forschung / Abteilung Biomolekulare Mechanismen
Directed Evolution of the DnaK Chaperone: Mutations in the Lid Domain Result in Enhanced Chaperone Activity
Translation of Title:Directed Evolution of the DnaK Chaperone: Mutations in the Lid Domain Result in Enhanced Chaperone Activity
Authors:Aponte, Raphael A.; Zimmermann, Sabine; Reinstein, Jochen
Date of Publication (YYYY-MM-DD):2010-05-28
Title of Journal:Journal of Molecular Biology
Journal Abbrev.:J. Mol. Biol.
Issue / Number:1
Start Page:154
End Page:167
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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.
Free Keywords:chaperone; DnaK; directed evolution; protein folding; lid domain
Last Change of the Resource (YYYY-MM-DD):--
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen
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