TABLE 3.
Interaction of Hda with the wild-type β and βE202K clamp proteins
| Interactiona | Expt | KD (nM) | ka (M−1s−1) | Kd (s−1) | Avg (range)b |
|
|---|---|---|---|---|---|---|
| KD 1 | KD 2 | |||||
| Hisβ-Hda | 1 | KD1, 5.47 | 2.23 × 104 | 1.22 × 10−4 | 6.38 (1.82) | 36.2 (0.20) |
| KD2, 36.1 | 1.66 × 105 | 6.01 × 10−3 | ||||
| 2 | KD1, 7.29 | 2.13 × 104 | 1.56 × 10−4 | |||
| KD2, 36.3 | 1.71 × 105 | 6.21 × 10−3 | ||||
| HisβE202K-Hda | 1 | KD1, 8.26 | 2.00 × 104 | 1.65 × 10−4 | 8.44 (0.35) | 50.5 (6.30) |
| KD2, 53.6 | 1.25 × 105 | 6.72 × 10−3 | ||||
| 2 | KD1, 8.61 | 1.80 × 104 | 1.55 × 10−4 | |||
| KD2, 47.3 | 1.28 × 105 | 6.08 × 10−3 | ||||
a
The indicated His6-tagged clamp protein (ligand) was attached to the SPR sensor surface using penta-His antibody (Qiagen), while the untagged Hda protein (analyte) was flowed over the sensor surface. Interactions were analyzed using the two-site binding model. Instead of χ2 (goodness of fit), the ClampXP 3.50 software provides residual sum-of-squares (goodness of fit), which were each <10% of the respective Rmax, confirming the specificity of each interaction.
b
Average of 2 independent experiments, each examining 5 different concentrations (60 to 960 nM) of analyte (Hda) ± range.