Figure 6.

Fluorescence anisotropy assay of peptide binding to wt and I462T DnaK. F-APPY (35 nM) fluorescein-labeled peptide was incubated with the indicated concentrations of wt (■) or I462T (⧫) DnaK overnight to achieve equilibrium. Fluorescence polarization was determined at 25°C with excitation at 490 nm and emission at 535 nm. Data were fitted to a quadratic single-site binding equation to determine the K_d of DnaK for peptide, which is shown in the box. Data is expressed as fraction of DnaK bound to F-APPY. At saturation, the maximum polarization for wt was approximately 240 polarization milliunits, whereas the maximum for I462T was approximately 205 polarization milliunits.