Electrostatic Interactions between Peptides and the Molecular Chaperone DnaK

The molecular chaperone DnaK prevents intracellular protein misfolding and aggregation by transiently binding with newly synthesized polypeptides and protein folding intermediates. DnaK preferentially binds to peptides with basic residues (Arg/Lys) present on the outside of a hydrophobic core. The electrostatic contribution toward DnaK/peptide binding was determined by measuring the dissociation constant of DnaK complexes with two fluorescein-labeled peptides (f-NRLLLTG and f-NALLLTG) using fluorescence anisotropy. The measured dissociation constants, Kd, differ significantly at low ionic strength: at 20 mM phosphate buffer, Kd for DnaK and f-NRLLLTG is 0.2 μM while that for DnaK and f-NALLLTG is 1 μM. This difference, attributed to stronger Coulombic binding in the case of f-NRLLLTG, vanishes at high ionic strength due to electrostatic screening. For f-NRAAATG, no interaction with DnaK was apparent, showing that hydrophobic interactions are essential in chaperone/peptide binding. The electrostatic contribution to the interaction between DnaK and NRLLLTG is interpreted