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Fine Tuning of a Biological Machine: DnaK Gains Improved Chaperone Activity by Altered Allosteric Communication and Substrate Binding
Author(s) -
Schweizer Regina S.,
Aponte Raphael A.,
Zimmermann Sabine,
Weber Annika,
Reinstein Jochen
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000786
Subject(s) - allosteric regulation , luciferase , mutant , chaperone (clinical) , atp hydrolysis , protein folding , alanine , biochemistry , biophysics , directed evolution , allosteric enzyme , chemistry , biology , enzyme , amino acid , atpase , medicine , transfection , pathology , gene
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.