TABLE 1.
Kinetic and thermodynamic parameters obtained from the analysis of the pseudo-first order binding experiments between site-directed variants of N-SH2 and Gab2448–460 wild-type and Gab2448–460 M457A.
| Gab2448–460 |
Gab2448–460 M457A |
||||||||
| N-SH2 variant | kon (μM–1 s–1) | koff (s–1) | KD (μM) | ΔΔGeq (kcal mol–1) | kon (μM–1 s–1) | koff (s–1) | KD (μM) | ΔΔGeq (kcal mol–1) | ΔΔΔ G |
| WT | 19.5 ± 1.0 | 3.9 ± 0.2 | 0.2 ± 0.1 | 21.5 ± 2.4 | 80 ± 4 | 3.7 ± 0.4 | |||
| L7A | 15.7 ± 1.2 | 4.0 ± 0.2 | 0.3 ± 0.1 | 0.15 ± 0.03 | 19.3 ± 2.3 | 90 ± 4 | 4.6 ± 0.6 | 0.12 ± 0.01 | ** |
| I17V | 24.4 ± 1.7 | 4.5 ± 0.2 | 0.2 ± 0.1 | −0.05 ± 0.02 | 22.0 ± 2.3 | 90 ± 5 | 4.3 ± 0.5 | 0.09 ± 0.01 | ** |
| V22A | 11.2 ± 0.4 | 7.4 ± 0.4 | 0.7 ± 0.1 | 0.68 ± 0.04 | 17.7 ± 2.6 | 170 ± 9 | 9.7 ± 1.5 | 0.55 ± 0.05 | ** |
| T29S | 17.5 ± 0.5 | 4.3 ± 0.2 | 0.3 ± 0.1 | 0.13 ± 0.02 | 19.7 ± 2.7 | 80 ± 4 | 4.0 ± 0.6 | 0.05 ± 0.01 | ** |
| A30G | 29.2 ± 2.0 | 3.7 ± 0.2 | 0.1 ± 0.1 | −0.25 ± 0.02 | 17.1 ± 2.2 | 90 ± 4 | 5.1 ± 0.7 | 0.19 ± 0.10 | ** |
| T33S | 22.6 ± 2.4 | 3.6 ± 0.2 | 0.2 ± 0.1 | −0.12 ± 0.02 | 20.6 ± 2.9 | 80 ± 4 | 3.8 ± 0.6 | 0.02 ± 0.01 | ** |
| L35A | 7.0 ± 0.6 | 6.7 ± 0.3 | 1.0 ± 0.1 | 0.88 ± 0.09 | * | * | * | * | * |
| T48S | 6.4 ± 0.5 | 23 ± 1 | 3.7 ± 0.1 | 1.64 ± 0.36 | * | * | * | * | * |
| T50S | 15.6 ± 0.8 | 9.3 ± 0.5 | 0.6 ± 0.1 | 0.62 ± 0.05 | 20.2 ± 2.0 | 140 ± 7 | 6.8 ± 0.8 | 0.35 ± 0.10 | ** |
| L51A | 5.6 ± 1.1 | 18 ± 1 | 3.2 ± 0.2 | 1.56 ± 0.64 | 21.2 ± 3.3 | 160 ± 8 | 7.6 ± 1.2 | 0.41 ± 0.10 | 1.16 ± 0.42 |
| L59A | 6.7 ± 1.0 | 21 ± 1 | 3.2 ± 0.2 | 1.57 ± 0.52 | * | * | * | * | * |
| I60V | 9.1 ± 1.1 | 6.9 ± 0.3 | 0.8 ± 0.1 | 0.76 ± 0.10 | 15.2 ± 1.5 | 80 ± 4 | 5.0 ± 0.6 | 0.18 ± 0.02 | 0.58 ± 0.16 |
| I62V | 15.5 ± 1.4 | 4.5 ± 0.2 | 0.3 ± 0.1 | 0.21 ± 0.03 | 18.4 ± 1.5 | 120 ± 6 | 6.7 ± 0.6 | 0.34 ± 0.10 | ** |
| P74A | 48.7 ± 1.5 | 27 ± 1 | 0.6 ± 0.1 | 0.59 ± 0.04 | 22.5 ± 1.9 | 60 ± 3 | 2.6 ± 0.3 | −0.20 ± 0.02 | 0.79 ± 0.12 |
| L75A | 8.1 ± 1.1 | 6.1 ± 0.3 | 0.8 ± 0.1 | 0.76 ± 0.11 | 18.8 ± 3.2 | 110 ± 5 | 5.7 ± 1.0 | 0.25 ± 0.03 | 0.51 ± 0.17 |
| T76S | 16.9 ± 1.3 | 5.3 ± 0.3 | 0.3 ± 0.1 | 0.26 ± 0.03 | 19.6 ± 2.6 | 100 ± 5 | 5.0 ± 0.7 | 0.17 ± 0.02 | ** |
| V80A | 16.0 ± 1.2 | 4.4 ± 0.2 | 0.3 ± 0.1 | 0.19 ± 0.03 | 22.3 ± 5.4 | 130 ± 6 | 5.6 ± 1.4 | 0.24 ± 0.02 | ** |
| V81A | 12.0 ± 1.4 | 4.5 ± 0.2 | 0.4 ± 0.1 | 0.37 ± 0.05 | 20.5 ± 3.8 | 90 ± 5 | 4.5 ± 0.9 | 0.11 ± 0.01 | ** |
| L99A | 8.2 ± 1.7 | 160 ± 10 | 20 ± 0.2 | 2.59 ± 0.30 | * | * | * | * | * |
| V101A | 13.4 ± 0.4 | 7.0 ± 0.4 | 0.5 ± 0.1 | 0.55 ± 0.06 | 14.2 ± 1.7 | 170 ± 9 | 12 ± 1 | 0.68 ± 0.10 | ** |
| L104A | 16.5 ± 0.2 | 3.5 ± 0.2 | 0.2 ± 0.1 | 0.05 ± 0.02 | 22.1 ± 1.4 | 110 ± 6 | 5.2 ± 0.4 | 0.19 ± 0.02 | ** |
*Binding was abolished by this mutation. **The absolute value of calculated ΔΔΔG was below 0.4 kcal mol–1 and was excluded from analysis.