Table 1. ITC measurements for complex formation between Scd2 constructs and Cdc42.GTP or Cdc42.GDP.
All measurements were performed using 25 mM Hepes, pH 7.5, 50 mM NaCl, 2 mM MgCl2 and 2 mM DTT at 18 °C. Scd2 constructs were used at concentrations in the range 300–500 μM, and were titrated into Cdc42.GTP, Cdc42.GDP or Cdc42.GTPΔ14 at concentrations between 30 and 50 μM. Kd is given in units of 10−6 M, ΔH and TΔS are given in kcal/mol (1 kcal/mol≡4.184 kJ/mol). The stoichiometry of complex formation for each binding site is N=1.0±0.1 for titrations with Cdc42.GDP and N=1.0±0.25 for titrations with Cdc42.GTP (Cdc42Gln61→Leu generally contained 75–90% GTP, as determined by reverse-phase HPLC).
| Kd1 | ΔH1 | TΔS1 | Kd2 | ΔH2 | TΔS2 | |
|---|---|---|---|---|---|---|
| Titrated into Cdc42.GTP | ||||||
| Scd2-(1–536) | 0.021±0.009 | −3.99±0.06 | 6.30±0.34 | 3.01±1.11 | 0.84±0.15 | 8.23±0.12 |
| Scd2-(1–266) | 0.028±0.003 | −3.87±0.02 | 6.20±0.09 | 2.71±0.41 | 0.83±0.04 | 8.25±0.04 |
| Scd2-(275–536) | No binding | |||||
| Scd2-(1–86) | 3.00±1.01 | −2.35±0.23 | 5.03±0.03 | |||
| Scd2-(110–266) | 2.93±0.40 | 1.91±0.01 | 9.28±0.09 | |||
| Scd2-(110–536) | 4.06±0.57 | 2.67±0.54 | 9.85±0.63 | |||
| Titrated into Cdc42.GDP | ||||||
| Scd2-(1–536) | No binding | |||||
| Scd2-(1–266) | No binding | |||||
| Titrated into Cdc42.GTPΔ14 | ||||||
| Scd2-(1–536) | 0.021±0.01 | −5.24±0.04 | 4.97±0.15 | 6.21±0.32 | 1.04±0.19 | 7.97±0.3 |