Table 2.
Binding affinities of GDF-5 and BMP-2 to immobilized BMPR-IB variants
| GDF-5 | BMP-2 | |||
|---|---|---|---|---|
| KD (nM)a | ΔΔG (kcal mol−1)b | KD (nM)a | ΔΔG (kcal mol−1)b | |
| BMPR-IB | 1.3±0.55 | — | 4.8±1.80 | — |
| F66A | ⩾1000c | ⩾4.0 | n.b.d | ⩾4.5 |
| Q67A | 8.2±2.98 (6.3 × ) | 1.1 | 21.7±11.31 (4.5 × ) | 0.9 |
| H22S/H23G | 4.1±1.88 (3.2 × ) | 0.7 | 7.2±1.21 (1.5 × ) | 0.2 |
| aThe apparent binding constant KD was derived from calculating KD=koff/kon. Numbers in parentheses represent the relative change compared with wild-type BMPR-IB. | ||||
| bCalculated using ΔΔG=(−RTlnKD)wt−(−RTlnKD)var with R=1.98 cal mol−1 K−1 and T=293.15 K. Values ⩾2.0 kcal mol−1 identify a hot spot of binding. | ||||
| cThe apparent KD was estimated from the dose dependency of equilibrium binding and presents the lower limit due to technical limitations of the BIAcore2000 system. | ||||
| dNo binding above background levels could be detected, from the highest analyte concentration applicable in the analysis, the binding affinity was estimated to be ⩾10 μM. | ||||