Table II.
Kinetic and Thermodynamic Values for the YmdB-RNase III Interaction, and the Effect of Specific Mutationsa
| YmdB | RNase III | ka (M−1s−1)b | kd (s−1)b | KD (nM) | ΔGSPR (kcal/mol) | ΔΔGSPR (kcal/mol) | ΔΔGFoldX (kcal/mol)c | ΔΔGRobetta (kcal/mol)# |
|---|---|---|---|---|---|---|---|---|
| WT | WT | 1.0 × 105 | 6.1 × 10−3 | 61 | −9.9 | – | – | – |
| R40A | WT | 59.1 | 6.7 × 10−5 | 1100 | −8.1 | +1.8 | +2.3 | +2.5 |
| WT | D128A/D128′A | 6.4 × 104 | 0.37 | 5800 | −7.2 | +2.7 | +1.5 | +0.4 |
Proteins were purified as described in Supporting Information SI-3. Measurements were performed at room temperature in a pH 7.5 buffer containing 150 mM NaCl (see Supporting Information SI-3).
The χ2 values for the global fits ranged from 1.1 to 2.2.
An ensemble average was calculated over all the representative structures for the three restrained dockings [see Eq. (3) in Supporting Information SI-1]. The standard deviation of the FoldX values is 0.8 kcal/mol for the R40A YmdB mutant, and 0.3 kcal/mol for the D128A/D128′A RNase III double mutant. The standard deviation of the Robetta values is 0.3 and 0.2 kcal/mol, respectively. The value for the D128A/D128′A double mutant was approximated as the sum of the values calculated for the two heterodimer forms: D128A/WT and WT/D128′A [see Eq. (2) in Supporting Information SI-1].