Gephyrin binding enthalpies and binding entropies of GlyR β-loop variants determined by ITC
| Parameter | β-Loop variant | Two-site model9 | One-site model10 | |
|---|---|---|---|---|
| High-affinity site | Low-affinity site | |||
| ΔH [kcal/mol]1 | βL-wt (pH 8.0)3 | –19.2 ± 1.6 | –2.1 ± 0.3*** | – |
| βL-LO4 | – | – | –11.8 ± 2.5 | |
| βL-Core5 | – | – | –11.0 ± 0.6*** | |
| βL-HI6 | – | – | –21.5 ± 0.3*** | |
| βL-D407P/F408G7 | – | – | –13.8 ± 3.2 | |
| βL-wt (pH 7.4)8 | –14.2 ± 1.9 | –4.9 ± 0.9 | – | |
| ΔS [cal/mol * K]2 | βL-wt (pH 8.0)3 | –28.4 ± 5.3 | 19.5 ± 1.4 | – |
| βL-LO4 | – | – | –6.7 ± 3.5 | |
| βL-Core5 | – | – | –11.2 ± 2.0 | |
| βL-HI6 | – | – | –40.3 ± 1.1 | |
| βL-D407P/F408G7 | – | – | –21.7 ± 11 | |
| βL-wt (pH 7.4)8 | –14.2 ± 6.3 | 8.3 ± 4.2 | – | |
Mean values and SEM from three or more independent measurements.
↵ 1Binding enthalpy (ΔH in kcal/mol).
↵ 2Binding entropy (ΔS in cal/mol * K).
↵ 3GlyR β-loop residues 378–426, pH 8.0.
↵ 4GlyR β-loop residues 378–413.
↵ 5GlyR β-loop residues 394–413.
↵ 6GlyR β-loop residues 394–426.
↵ 7GlyR β-loop residues 378–426 with substitution D407P and F408G.
↵ 8GlyR β-loop residues 378–426, pH 7.4.
↵ 9Binding isotherm fitted to a two-site interaction with gephyrin.
↵ 10Binding isotherm fitted to a one-site model; Data were compared using an unpaired two-tailed t-test: *** p = 0.0005 ΔH of βL-wt high-affinity site n = 3 vs. low-affinity site n = 3; *** p = 0.0001 ΔH of βL-wt high-affinity site n = 3 vs. βL-Core n = 9; *** p < 0.0001 ΔH of βL-Core n = 9 vs. βL-HI n = 4; p = 0.6921 ΔH of βL-Core n = 9 vs. βL-LO n = 5.