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  Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding

Schweizer, R., Aponte, R. A., Zimmermann, S., Weber, A., & Reinstein, J. (2011). Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding. ChemBioChem: A European Journal of Chemical Biology, 12(10), 1559-1573. doi:10.1002/cbic.201000786.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-1EF5-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-1EF6-5
Genre: Journal Article
Alternative Title : Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding

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ChemBioChem_12_2011_1559.pdf (Any fulltext), 617KB
 
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 Creators:
Schweizer, Regina1, Author              
Aponte, Raphael A., Author
Zimmermann, Sabine1, Author              
Weber, Annika1, 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: chaperones; directed evolution; protein engineering; protein folding; transient kinetics
 Abstract: DnaK is a member of the Hsp70 family of molecular chaperones. This molecular machine couples the binding and hydrolysis of ATP to binding and release of substrate proteins. The switches that are involved in allosteric communication within this multidomain protein are mostly unknown. Previous insights were largely obtained by mutants, which displayed either wild−type activity or reduced folding assistance of substrate proteins. With a directed evolution approach for improved folding assistance we selected a DnaK variant characterized by a glycine to alanine substitution at position 384 (G384A); this resulted in a 2.5−fold higher chaperone activity in an in vitro DnaK−assisted firefly luciferase refolding assay. Quantitative biochemical characterization revealed several changes of key kinetic parameters compared to the wild type. Most pronounced is a 13−fold reduced rate constant for substrate release in the ATP−bound state, which we assume, in conjunction with the resulting increase in substrate affinity, to be related to improved chaperone activity. As the underlying mechanistic reason for this change we propose an altered interface of allosteric communication of mutant G384A, which is notably located at a hinge position between nucleotide and substrate binding domain

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Language(s): eng - English
 Dates: 2010-12-232011-06-082011-07-04
 Publication Status: Published in print
 Pages: 15
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Degree: -

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Title: ChemBioChem : A European Journal of Chemical Biology
  Other : ChemBioChem
Source Genre: Journal
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Publ. Info: Weinheim, Germany : Wiley-VCH
Pages: - Volume / Issue: 12 (10) Sequence Number: - Start / End Page: 1559 - 1573 Identifier: ISSN: 1439-4227
CoNE: https://pure.mpg.de/cone/journals/resource/110978984568897_1