Figure 4.

Functional assays reveal that Hsc70(1–508) (NBD–BETA) behaves similarly to wt-Hsc70. A, HLA tracer binding in the ADP and ATP states, as measured by fluorescence polarization. Dark blue, NBD–BETA ADP; light blue, NBD–BETA ATP; black, wt-Hsc70 ADP; gray, wt-Hsc70 ATP. Note that the wt-Hsc70 curves could not reach saturation because of limitations on protein solubility. B, DJA2 stimulation of ATP turnover, as measured by malachite green assays. Blue, NBD–BETA; black, wt-Hsc70. C, refolding of denatured luciferase. Blue, NBD–BETA; black, wt-Hsc70; gray, no Hsc70 control. Note that DJA2 will first stimulate and then inhibit ATP turnover, showing a maximal stoichiometry. D, the ATPase activity of neither wt-Hsc70 nor NBD–BETA can be stimulated by peptide substrates. Red, DnaK by NR; black, wt-Hsc70 by TAU1; blue, NBD–BETA by TAU1.