Table 3.
Effect of nucleotides on the ARE-binding affinity of select Hsp70 proteins
Apparent equilibrium dissociation constants for the indicated proteins binding to the Fl-ARE[34] RNA substrate were resolved using fluorescence anisotropy-based assays as described in Fig. 3 and are given as the mean ± S.D. from n independent experiments.
| Protein | Nucleotidea | Kd app | n |
|---|---|---|---|
| nm | |||
| His6-Hsp70wt | None | 11.7 ± 2.7 | 4 |
| ATP | 10.4 ± 0.9 | 3 | |
| ADP | 12.6 ± 2.2 | 3 | |
| AMP-PNP | 14.4 ± 1.4 | 3 | |
| His6-Hsp70(1–385) | None | 31.8 ± 2.0 | 4 |
| ATP | 26.3 ± 4.1 | 3 | |
| ADP | 22.7 ± 4.5 | 4 | |
| AMP-PNP | 35.7 ± 6.6 | 3 |
a Nucleotides were added to 2 mm where indicated. All reactions contained 2 mm MgCl2 to permit formation of Mg2+–nucleotide complexes.