Document Detail


Fine Tuning of a Biological Machine: DnaK Gains Improved Chaperone Activity by Altered Allosteric Communication and Substrate Binding.
MedLine Citation:
PMID:  21656889     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Regina S Schweizer; Raphael A Aponte; Sabine Zimmermann; Annika Weber; Jochen Reinstein
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-6-8
Journal Detail:
Title:  Chembiochem : a European journal of chemical biology     Volume:  -     ISSN:  1439-7633     ISO Abbreviation:  -     Publication Date:  2011 Jun 
Date Detail:
Created Date:  2011-6-9     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100937360     Medline TA:  Chembiochem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Affiliation:
Department for Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany), Fax: (+49) 6221-486-585.
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