Table 2.
Affinities and thermodynamic values of homing endonuclease–DNA-binding events
| Protein | Site | KD | ΔH | ΔS | −TΔS | ΔG |
|---|---|---|---|---|---|---|
| nM | kcal/mol | cal/mol/deg | kcal/mol | kcal/mol | ||
| I-PpoI | WT | 17 ± 1.7 | −35.5 ± 0.29 | −81.6 ± 0.87 | 24.7 | −10.8 |
| I-PpoI | LL | 87 ± 8.8 | −35.1 ± 0.57 | −83 ± 3.0 | 25.3 | −9.8 |
| I-PpoI | RR | 23 ± 1.6 | −38.8 ± 0.26 | −93.1 ± 2.4 | 28.2 | −10.6 |
| I-AniI | WT | 96 ± 10 | 11.2 ± 1.1 | 69.3 ± 3 | −21.0 | −9.8 |
| I-AniI | WT-OPT | 8 ± 1 | 6.6 ± 0.7 | 59.0 ± 3 | −17.9 | −11.3 |
| I-MsoI | WT | 21 ± 5.0 | 12.7 ± 0.27 | 77 ± 1.1 | −23.3 | −10.6 |
| I-MsoI | LL | 6 ± 1.4 | 16.4 ± 0.20 | 91.7 ± 0.36 | −27.8 | −11.4 |
| I-MsoI | RR | 17 ± 6.0 | 14.4 ± 0.40 | 83.0 ± 0.64 | −25.2 | −10.8 |
| I-MsoI | MIS | 760 ± 53 | 21.2 ± 0.42 | 98 ± 2.7 | −29.7 | −8.4 |
| I-MsoI redesigned | MIS | 46 ± 6.5 | 34.2 ± 0.42 | 146.5 ± 0.7 | −44.4 | −10.2 |
| I-MsoI redesigned | WT | 243 ± 20 | 37.1 ± 4 | 152.7 ± 6 | −46.3 | −9.2 |
| I-SspI (E11Q)a | WT | 350 ± 30 | −23.3 ± 3 | −47.3 ± 4 | 14.3 | −9.0 |
| I-HmuIb | WT | (30) | Endothermic | – | – | – |
a‘I-SspI’ corresponds to a catalytically inactive mutant (E11Q) of the I-Ssp8603I homing endonuclease, containing an alteration of an active-site putative metal-binding residue. In addition, the enzyme contains a second mutation on its surface far from the DNA-binding interface (F55K) to improve its solubility. The resulting affinity and ΔGbinding therefore may not reflect the wild-type enzyme.
bI-HmuI displays complex behavior in ITC experiments that is not reliably modeled mathematically (Figure 2). The reaction is strongly endothermic, and the Kd for that binding event has been estimated at 30 nM.