Table 3.
Calorimetric binding data for L3MBTL3 alanine mutants and native RBPJ
| L3MBTL3 | K (M−1) | K d (μM) | ΔG° (kcal/mol) | ΔH° (kcal/mol) | –TΔS° (kcal/mol) | ΔΔG° (kcal/mol) |
|---|---|---|---|---|---|---|
| WT | 1.1 ± 0.1 × 106 | 0.92 | –8.3 ± 0.1 | –13.1 ± 0.3 | 4.8 ± 0.3 | – |
| WT + DMSO | 1.1 ± 0.1 × 106 | 0.90ns | –8.3 ± 0.1 | –12.8 ± 0.6 | 4.5 ± 0.7 | 0.0 |
| N54A | 2.1 ± 0.6 × 106 | 0.47* | –8.6 ± 0.2 | –11.1 ± 0.9 | 2.5 ± 1.1 | -0.3 |
| V55A | 1.1 ± 0.2 × 106 | 0.87ns | –8.3 ± 0.1 | –13.6 ± 1.0 | 5.3 ± 1.1 | 0.0 |
| K56A + DMSO | 5.6 ± 1.8 × 105 | 1.8** | –7.8 ± 0.2 | –8.0 ± 1.2 | 0.2 ± 1.3 | 0.5ψ |
| K57A | 1.9 ± 0.4 × 105 | 5.3*** | –7.2 ± 0.2 | –11.9 ± 0.7 | 4.7 ± 0.7 | 1.1 |
| A58R | 1.9 ± 0.3 × 106 | 0.54** | –8.6 ± 0.1 | –14.0 ± 0.9 | 5.4 ± 0.9 | –0.3 |
| T59A | 7.6 ± 1.1 × 105 | 1.3* | –8.0 ± 0.1 | –9.9 ± 0.7 | 1.9 ± 0.8 | 0.3 |
| A60R | 2.4 ± 0.5 × 105 | 4.2*** | –7.3 ± 0.1 | –15.7 ± 0.3 | 8.4 ± 0.4 | 1.0 |
| T61A | 5.8 ± 0.6 × 105 | 1.7** | –7.9 ± 0.1 | –11.4 ± 0.6 | 3.5 ± 0.6 | 0.4 |
| T62A | 3.3 ± 0.5 × 106 | 0.30*** | –8.9 ± 0.1 | –13.7 ± 0.3 | 4.8 ± 0.4 | –0.6 |
| Δ62 | 1.0 ± 0.03 × 106 | 0.99ns | –8.2 ± 0.0 | –12.7 ± 0.7 | 4.5 ± 0.7 | 0.1 |
| T63A | NBD | — | — | — | — | — |
| W64A | NBD | — | — | — | — | — |
| M65A | 1.0 ± 0.3 × 106 | 1.0ns | –8.1 ± 0.2 | -12.4 ± 1.7 | 4.3 ± 1.9 | 0.2 |
| V66A | NBD | — | — | — | — | — |
| P67A | NBD | — | — | — | — | — |
| T68A | 8.7 ± 1.0 × 105 | 1.2ns | –8.1 ± 0.1 | –11.7 ± 0.7 | 3.6 ± 0.7 | 0.2 |
| A69R | 6.1 ± 0.7 × 105 | 1.63** | –7.9 ± 0.1 | –13.8 ± 1.1 | 5.9 ± 1.2 | 0.4 |
| Q70A | 1.1 ± 0.4 × 106 | 0.94ns | –8.3 ± 0.3 | –14.2 ± 0.3 | 5.9 ± 0.3 | 0.0 |
Constructs: L3MBTL3 (52–70) and RBPJ (53–474). All experiments were performed at 25°C. NBD represents no binding detected. Values are the mean of at least three independent experiments and errors represent the standard deviation of multiple experiments. ΔΔG° = ΔG°(mutant) – ΔG°(mutant). ψRelative to WT + DMSO value. K56A peptide required dissolution in a phosphate buffer with 5% DMSO. P-value determined from a two-tailed t-test (***P < 0.001, **P < 0.01, *P < 0.05 and ns, not significant), comparing WT L3MBTL3 to point mutants.