TABLE 3.
ITC binding constants and thermodynamic parameters
ITC experiments were performed at 22 °C in 25 mm Bis-tris propane buffer, pH 7.8, 100 mm NaCl, 5 mm Tris(2-carboxyethyl)phosphine. At the concentrations used in the ITC experiments, all proteins were monomeric as shown by AUC (Table I). Binding was dominated by favorable entropy changes, and the contribution of TΔS to ΔG was significantly higher than ΔH. The BMAL490(K537Q)-P2 interaction was entropically and enthalpically favored. N, number of binding sites (N = ligand/receptor). Except for the mBMAL1-P1 complexes, the mBMAL1 fragments were used as ligands. Reported values and S.D. are the mean of at least three independent titrations.
| Complex | N | KD | ΔH | TΔS |
|---|---|---|---|---|
| μm | kcal/mol | kcal/mol | ||
| mCRY1CCT-BMAL577 | 0.9 | 10.5 ± 2.3 | 2.6 | 9.2 |
| mCRY1CCT-BMAL490 | 0.8 | 18.9 ± 5.0 | 1.8 | 8.1 |
| mCRY1CCT-BMAL490(K537Q) | 0.8 | 9.3 ± 2.2 | 1.4 | 8.2 |
| mCRY2CCT-BMAL577 | 1.1 | 7.8 ± 1.2 | 1.0 | 8.4 |
| mCRY2CCT-BMAL490 | 1.1 | 9.5 ± 2.2 | 4.2 | 11.1 |
| P1-BMAL577 | 1.0 | 10.7 ± 2.6 | 1.1 | 8.3 |
| P1-BMAL490 | 0.8 | 10.6 ± 3.0 | 1.3 | 8.1 |
| P1-BMAL490(K537Q) | 1.0 | 9.6 ± 2.0 | 0.7 | 7.5 |
| P2-BMAL577 | 0.9 | 3.3 ± 1.0 | 0.5 | 8.0 |
| P2-BMAL490 | 0.9 | 7.7 ± 1.9 | 7.3 | 14.2 |
| P2-BMAL490(K537Q) | 1.0 | 2.9 ± 0.9 | −0.6 | 6.8 |