Fig. 3.

DnaK NBD substitution mutants are defective in direct interaction with Hsp90_Ec in vitro. (a) Bio-Layer Interferometry (BLI) was used to monitor the kinetics of association and dissociation between biotinylated Hsp90_Ec and DnaK wild-type as described in Materials and Methods. Representative curves are shown for numerous concentrations of DnaK as indicated. The single-exponential fit of the association step was used to obtain the response value (pm) for the plateau plotted in (b). (b) Steady-state analysis of the DnaK wild-type-Hsp90_Ec interaction. The response value (pm) for the plateau obtained in (a) is plotted vs. each DnaK concentration and fit as described in Materials and Methods. The K_d and Bmax for the Hsp90_Ec interaction with DnaK wild-type are 13.4 ± 3.3 µM and 0.12 ± 0.01 nm, respectively. (c) Curves showing association and dissociation of 50 µM DnaK wild-type (black) or mutant (colored) and biotinylated Hsp90_Ec using BLI. (d) Average plateau response value for the interaction between DnaK wild-type or mutant and biotinylated Hsp90_Ec. Data are plotted as mean ± SEM (n=2 or more) and are also presented in Supplemental Table S5. (e) Steady-state analysis of the DnaK_{Y145A,N147A,D148A}-Hsp90_Ec interaction as described in (b). The K_d and Bmax for the Hsp90_Ec interaction with DnaK_{Y145A,N147A,D148A} are 44.4 ± 7.5 µM and 0.10 ± 0.01 nm, respectively.