Table 2.
Thermodynamic parameters for anion binding to WT and mutant CLC-ec1. Thermodynamic parameters, Kd, ΔHo, TΔS° and ΔG° are determined as described in Table 1, and by fixing n=1 for WT, Y445A and S107P. We fixed n=2 for the E148A mutant since the affinity of Br−, NO3− and SCN− is not sufficient to allow for the independent determination of the number of binding sites.
| Protein | Anion | Kd (μM) | ΔH° (Kcal Mol−1) | TΔS° (Kcal Mol−1) | ΔG° (Kcal Mol−1) |
|---|---|---|---|---|---|
| WT | Br− | 2550±530 | −5.0±0.6 | −1.4±0.5 | −3.6±0.1 |
| NO3− | 13000±1500 | −6.9±1.2* | −4.3±1.2* | −2.6±0.1 | |
| SCN− | 9200±400 | −15.6±0.1* | −12.8±0.1* | −2.78±0.03 | |
| Y445A | Br− | no heat detected | |||
| NO3− | no heat detected | ||||
| SCN− | 6700±200 | −21.0±0.6* | −18.2±0.6* | −2.96±0.02 | |
| E148A | Br− | 84±16 | −3.4±0.9 | 2.2±1.0 | −5.6±0.5 |
| NO3− | 1400±300 | −5.1±0.5 | −1.2±0.6 | −3.9±0.1 | |
| SCN− | 5800±1200 | −7.7±1.6* | −4.7±1.5* | −3.1±0.1 | |
| S107P | Cl− | no heat detected | |||
| Br− | no heat detected | ||||
| NO3− | 3800±800 | −2.9±0.7 | 0.5±0.6 | −3.3±0.1 | |
| SCN− | 8000±1500 | −15.1±1.7* | −12.2±1.8* | −2.9±0.1 | |
*
These enthalpy and entropy values are only estimates since the binding reaction could not be driven to complete saturation. Each value represents the mean of three or more independent ITC experiments and the s.e.m. is reported as the error (see Supplementary Table 1 for the results of the individual experiments).