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          Institute: MPI für medizinische Forschung     Collection: jahrbuch_2011_archival     Display Documents



ID: 597779.0, MPI für medizinische Forschung / jahrbuch_2011_archival
Fine Tuning of a Biological Machine: DnaK Gains Improved Chaperone Activity by Altered Allosteric Communication and Substrate Binding
Translation of Title:Fine Tuning of a Biological Machine: DnaK Gains Improved Chaperone Activity by Altered Allosteric Communication and Substrate Binding
Authors:Schweizer, Regina S.; Aponte, Raphael A.; Zimmermann, Sabine; Weber, Annika; Reinstein, Jochen
Language:English
Date of Publication (YYYY-MM-DD):2011-07-04
Title of Journal:ChemBioChem
Journal Abbrev.:ChemBioChem
Volume:12
Issue / Number:10
Start Page:1559
End Page:1573
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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
Free Keywords:chaperones;
directed evolution;
protein engineering;
protein folding;
transient kinetics
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen
MPI für medizinische Forschung/Abteilung Biomolekulare Mechanismen/Molecular chaperones
Identifiers:LOCALID:7731
URI:http%3A%2F%2Fonlinelibrary.wiley.com%2Fdoi%2F10.10...
URI:http%3A%2F%2Fonlinelibrary.wiley.com%2Fdoi%2F10.10...
URI:http%3A%2F%2Fonlinelibrary.wiley.com%2Fdoi%2F10.10...
DOI:10.1002%2Fcbic.201000786
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