FIGURE 5.
ATP binding to NBD2 allows cooperative binding of ATP to the NBD1 in the presence of a substrate protein. A, shown is the percentage of protection against aggregation of thermally denatured MDH (6 μm) by increasing concentrations of T1T2 and T1N2. Experiments were performed at 47 °C in 50 mm Tris-HCl, pH 7.5, 20 mm MgCl2, 150 mm KCl, 2 mm DTT, and 2 mm ATP. Data corresponding to WT ClpB, T1, T2, and N2 indicate that they cannot form stable complexes with denatured MDH. B, aggregation of 6 μm MDH in the presence of 4 μm T1T2 (solid lines) or 12 μm T1N2 (dashed lines) at increasing ATP concentration is shown. Experiments were carried out as in A. Scattering values are normalized to controls obtained without ATP. C, extent of protection against aggregation of 6 μm MDH by 4 μm T1T2 or 12 μm T1N2 as a function of ATP concentration is shown. Experiments were carried out as described in B. Analysis of the data with the Hill equation gives Kd values of 61 ± 3 and 363 ± 12 μm and nH values of 3.7 ± 0.6 and 1.3 ± 0.1 for T1T2 and T1N2, respectively. Data are the means ± S.D. (n = 3).