Table 4.
Alanine substitutions in the DNA recognition helix of IscR decrease binding affinity for iscRB
| IscR variant | Binding affinity (Kd)a nM | Hill slope (n)b | Maximum anisotropy (Amax)c | % wild-type binding activity |
|---|---|---|---|---|
| wild-type | 24 ± 1d | 2.3 ± 0.8 | 0.19 ± 0.01 | 100 |
| S40A | 94 ± 3 | 1.5 ± 0.1 | 0.18 ± 0.01 | 26 |
| Y41A | >2000 | 1.3 ± 0.3 | 0.18 ± 0.01 | <1 |
| E43A | 21 ± 1 | 2.3 ± 0.2 | 0.19 ± 0.01 | 115 |
| Q44A | >600 | 1.3 ± 0.1 | 0.21 ± 0.01 | <4 |
| R59A | n.d.e | n.d. | n.d. | <1 |
a, b, c
The Binding affinity (Kd), Hill slope (n), and maximum anisotropy (Amax) for each variant protein with the iscRB site was determined from DNA binding isotherms (shown in Supplementary Fig. 2). Purified variant IscR proteins were incubated anaerobically with a DNA containing the iscRB site.
d
The given standard deviation is derived from fitting a four parameter Hill equation to the average of at least three independent binding experiments for each variant.
e
No observable binding at the IscR protein concentrations tested.