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

Aponte, R. A., Zimmermann, S., & Reinstein, J. (2010). Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity. Journal of Molecular Biology (London), 399(1), 154-167. doi:10.1016/j.jmb.2010.03.060.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-1FCA-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-1FCB-5
Genre: Journal Article
Alternative Title : Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity

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 Creators:
Aponte, Raphael A.1, Author              
Zimmermann, Sabine1, Author              
Reinstein, Jochen1, Author              
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1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: chaperone; DnaK; directed evolution; protein folding; lid domain
 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.

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Language(s): eng - English
 Dates: 2010-03-262009-10-192010-03-302010-04-082010-05-28
 Publication Status: Published in print
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 664603
DOI: 10.1016/j.jmb.2010.03.060
URI: http://www.ncbi.nlm.nih.gov/pubmed/20381501
Other: 7583
 Degree: -

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Title: Journal of Molecular Biology (London)
  Other : J Mol Biol
Source Genre: Journal
 Creator(s):
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Publ. Info: London : Academic Press
Pages: - Volume / Issue: 399 (1) Sequence Number: - Start / End Page: 154 - 167 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/954922646042