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. Author manuscript; available in PMC: 2021 Jul 30.
Published in final edited form as: Biochemistry. 2020 May 4;59(20):1946–1960. doi: 10.1021/acs.biochem.0c00320

Figure 5.

Figure 5.

KLR-70 binds DnaK with high affinity. (A) Representative direct binding isotherm generated from the native gel analysis of ADP-DnaK (5 μM) preequilibrated in the presence of variable concentrations (0 – 50 μM) of KLR-70. Additional information on the generation and analysis of these data can be found in the Materials and Methods and Figure S2. (B) Representative direct-binding isotherm generated from changes in the anisotropy of KLR-70-A-AF488 (10 nM) preequilibrated in the presence of variable concentrations (0 – 100 μM) of ADP-DnaK. (C) Representative competitive binding isotherm generated from changes in the steady-state anisotropy of KLR-70-C-AF488 (10 nM) preequilibrated with ADP-DnaK (10 μM) and variable amounts of KLR-70 (1 – 1000 μM). Curve fitting yields dissociation constants of (A) 158 ± 70 nM, (B) 1.73 ± 0.37 μM and (C) 889 ± 386 nM. All dissociation constants are reported as avg ± standard error for (A) n = 3 or (B and C) n = 4 independent experiments.