Table 1.
Comparison of properties of the wild-type and designed AcPh proteins
| AcPh-wt | AcPh-des | |
|---|---|---|
| MWtheor, kDa | 12.2 | 12.2 |
| MWAUC, kDa | 11.2 ± 0.9 | 12.2 ± 0.1 |
| Stability | ||
| Tm (CD or DSC), °C | 57.0 ± 0.5; 57.6 ± 0.1 | 66.2 ± 0.5; 66.3 ± 0.1 |
| ΔH (CD or DSC), kJ/mol | 333 ± 12; 328 ± 9 | 365 ± 16; 343 ± 8 |
| ΔΔG (CD or DSC), kJ/mol | 0 | 9.3 ± 1; 8.3 ± 0.5 |
| Activity | ||
| 25 °C | ||
| Km, M | (1.0 ± 0.2)·10−4 | (2.3 ± 0.2)·10−4 |
| kcat, s−1 | (1.0 ± 0.1)·103 | (0.9 ± 0.1)·103 |
| 40 °C | ||
| Km, M | (1.2 ± 0.1)·10−4 | (2.2 ± 0.2)·10−4 |
| kcat, s−1 | (1.6 ± 0.1)·103 | (1.6 ± 0.1)·103 |
| 55 °C | ||
| Km, M | (3.0 ± 0.2)·10−4 | (4.0 ± 0.3)·10−4 |
| kcat, s−1 | (2.4 ± 0.2)·103 | (2.4 ± 0.2)·103 |
MWtheormolecular mass calculated from the amino acid sequence. MWAUC molecular mass in solution was obtained from the analytical ultracentrifugation experiments as described [data for AcPh-wt from Strickler et al. (7); actual experimental data for AcPh-des, Cdc42-wt, CDC42-des1, and Cdc42-des2 are presented in Fig. S2]. Tm (CD/DSC) and ΔH (CD/DSC) are the values for the transition temperature and enthalpy of unfolding, respectively, obtained from the analysis of CD or DSC by using a two-state model and ΔCp of 4.5 kJ/(mol·K) as shown in Fig 3 A and B. ΔΔG (CD/DSC) values of Gibbs energy at 57° C; Km and kcat were obtained from the fit of the initial velocity versus concentration of benzoylphosphate as a substrate by using Michaelis-Menten relationship (see Fig. S4).