The binding affinities between Hsp90 and Cdc37 as well as those of all mutants were determined by ITC (MicroCal iTC200). Proteins in titration cells were prepared at a concentration of 50 μM in assay buffer [20 mM tris-HCl and 150 mM NaCl (pH 7.4)], and proteins in syringes were prepared at a concentration of 300 μM in the same assay buffer. Intervals of 180 s and a stirring speed of 1000 rpm were used for the entire injection procedure, which involved a total of 19 injections. In addition, the first 0.5 μl of the ligand solution was titrated to prevent initial interference. All the data obtained from the experiment were analyzed by the Origin software package to determine binding parameters, including the association constant (Ka = 1/Kd), enthalpy value (ΔH), and entropy value (ΔS).

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