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; Adenosine Triphosphate; Amino Acid Substitution; Animals; Directed Molecular Evolution; Escherichia coli; Escherichia coli Proteins; Fireflies; HSP70 Heat-Shock Proteins; Luciferases, Firefly; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protein Refolding
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Phenotype of	Taxon Information	Genotype Information (if known)	Condition Information	OMP ID	OMP Term Name	ECO ID	ECO Term Name	Notes	Status
a mutation or genetic difference within a strain	Taxon: Escherichia coli Strain: K-12 Substrain: NCBI Taxon ID: 83333	Genotype of Reference Strain: DnaK Genotype of Experimental Strain : DnaKG384A	Reference Condition:					Glycine to Alanine at position 384 mutation. There is a 13 fold decrease rate for substrate release in ATP bound state and increase in substrate affinity. with an overall 2.5 fold higher chaperon activity, Table 1.
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