PMID:21656889
Citation |
Schweizer, RS, Aponte, RA, Zimmermann, S, Weber, A and Reinstein, J (2011) Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding. Chembiochem 12:1559-73 |
---|---|
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. |
Links |
PubMed Online version:10.1002/cbic.201000786 |
Keywords |
Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Amino Acid Substitution; Animals; Directed Molecular Evolution/methods; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fireflies/metabolism; HSP70 Heat-Shock Proteins/genetics; HSP70 Heat-Shock Proteins/metabolism; Luciferases, Firefly/metabolism; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protein Refolding |
edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<protect><annotationlinks/></protect>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.