TABLE 4.
DNA binding affinity of wild-type and mutant enzymes
The dissociation constants (KD) were determined by surface plasmon resonance using BIACORE 2000 with a 16-nt DNA oligomer (5′-Biotin-AGT GGT TGG TGT CGG G-3′).
| Proteins | 16-mer ssDNA |
|
|---|---|---|
| KD | Relative KD | |
| nm | ||
| WT RNase II | 8.7 ± 0.7 | 1.0 |
| D201N | 15.9 ± 2.4 | 1.8 |
| Y313F | 6.9 ± 0.3 | 0.8 |
| Y313A | ≫100 | ≫100 |
| E390A | 75.7 ± 8.8 | 8.7 |
| D201N/Y313F | 6.4 ± 0.5 | 0.7 |
| D201N/E390A | 12.8 ± 2 | 1.5 |
| Y313F/E390A | 12.0 ± 0.4 | 1.4 |
| D201N/Y313F/E390A | 10.7 ± 0.3 | 1.2 |
| R500A | 14.2 ± 0.2 | 1.6 |
| E542A | 6.1 ± 0.3 | 0.7 |