DnaK is a 70 kilodalton heat shock protein and molecular chaperone from Escherichia coli with an N-terminal nucleotide binding domain and C-terminal substrate binding domain. During chaperone function DnaK occupies at least two conformational states characterized by distinct biochemical properties and interdomain distances. Here we develop a method for fluorescent double labeling of DnaK, creating a reagent that reports DnaK’s conformational shift via changes in Förster resonance energy transfer. We show that selective labeling can be accomplished by identifying a pair of cysteine residues whose local environments render one thiol several-fold more reactive than the other. Reactivity of cysteines introduced at various positions on the surface of DnaK was assayed using two different fluorescent dyes and two distinct assays. The fluorescent compound 7-diethylamino-3-(4’-maleimidylphenyl)-4- methylcoumarin reacted threefold faster to DnaK single-cysteine variant T136C/C15S than to S423C/C15S (rate constants of 0.124 ± 0.003 s-1 and 0.0433 ± 0.0005 s-1, respectively). This difference in reactivity was predictive of relative reactivity to Alexa Fluor 555 maleimide, for which we observed a twentyfold difference in reactivity between T136C and S423C (rate constants of 0.029 ± 0.003 s-1 and 0.00144 ± 0.00004 s-1, respectively). We prepared a variant of DnaK that contains two cysteines at positions 136 and 423 and showed that Alexa Fluor 594 maleimide reacts 13-fold faster to T136C in the nucleotide binding domain than to S423C in the substrate binding domain (rate constants of 0.048 ± 0.005 s-1 and 0.0036 ± 0.0006 s-1, respectively). Limited proteolysis demonstrated that DnaK T136C/S423C/C15S is capable of undergoing a nucleotide-dependent conformational change, suggesting it is functionally active.
We overcame low solubility of double-labeled DnaK T136C/S423C/C15S by optimizing buffer conditions and labeling with donor and acceptor fluorophores sequentially in the same reaction vessel. Our improved labeling method allowed us to make doublelabeled protein that reports DnaK’s conformational change with a 16% decrease in Alexa Fluor 555 donor fluorescence upon addition of adenosine triphosphate. We use Förster resonance energy transfer together with small angle x-ray scattering and limited proteolysis to show that therapeutically applicable heat shock protein 70 activity modulators interfere with DnaK’s conformational shift.
