Table 1. Kinetic parameters for Val→Thr and Val→Ala mutants of D48G-SH3.
| Proteins | k‡–U*, s-1 | m‡–U†, kcal·mol-1·M-1 | k‡–F,* s-1 | m‡–F†, kcal·mol-1·M-1 | ΔΔG‡–U§, kcal·mol-1 | ΔΔG‡–F§ kcal·mol-1 | ΔΔGF–U kcal·mol-1 | Φ‡–U¶ |
|---|---|---|---|---|---|---|---|---|
| D48G∥ | 63.63 ± 1.20 | –0.78 ± 0.01 | 0.010 ± 0.000 | 0.47 ± 0.00 | – | – | – | – |
| V9A | 51.59 ± 1.84 | –0.76 ± .01 | 0.009 ± 0.009 | 0.43 ± 0.12 | – | – | – | – |
| V9T | 52.52 ± 3.08 | –0.73 ± 0.03 | 0.297 ± 0.054 | 0.45 ± 0.02 | –0.11 ± 0.04 | 2.00 ± 0.11 | –2.12 ± 0.11 | 0.05 ± 0.05 |
| V23A | 16.45 ± 0.67 | –0.77 ± 0.02 | 0.055 ± 0.008 | 0.46 ± 0.02 | –0.80 ± 0.03 | 1.01 ± 0.09 | –1.81 ± 0.09 | 0.44 ± 0.05 |
| V23T | 21.87 ± 0.63 | –0.78 ± 0.01 | 0.042 ± 0.006 | 0.45 ± 0.02 | –0.63 ± 0.02 | 0.85 ± 0.08 | –1.48 ± 0.09 | 0.43 ± 0.06 |
| V44A | 2.75 ± 0.16 | –0.91 ± 0.04 | 0.117 ± 0.005 | 0.37 ± 0.01 | –1.86 ± 0.04 | 1.46 ± 0.02 | –3.32 ± 0.04 | 0.56 ± 0.02 |
| V44T | 1.11 ± 0.08 | –0.83 ± 0.05 | 0.065 ± 0.004 | 0.48 ± 0.01 | –2.40 ± 0.04 | 1.11 ± 0.04 | –3.51 ± 0.06 | 0.68 ± 0.02 |
| V46A | 11.41 ± 0.36 | –0.89 ± 0.01 | 0.008 ± 0.002 | 0.37 ± 0.04 | –1.02 ± 0.02 | –0.13 ± 0.15 | –0.89 ± 0.15 | 1.15 ± 0.26 |
| V46T | 8.98 ± 0.27 | –0.86 ± 0.01 | 0.002 ± 0.000 | 0.46 ± 0.02 | –1.16 ± 0.02 | –0.90 ± 0.00 | –0.20 ± 0.02 | 5.62 ± 0.58 |
| V53A | 8.53 ± 0.62 | –0.97 ± 0.04 | 0.054 ± 0.007 | 0.43 ± 0.02 | –1.19 ± 0.04 | 1.00 ± 0.08 | –2.19 ± 0.09 | 0.54 ± 0.05 |
| V53T | 2.79 ± 0.18 | –0.86 ± 0.04 | 0.079 ± 0.006 | 0.47 ± 0.01 | –1.85 ± 0.04 | 1.22 ± 0.05 | –3.07 ± 0.06 | 0.60 ± 0.02 |
| V58A | 40.05 ± 1.32 | –0.98 ± 0.02 | 0.404 ± 0.027 | 0.41 ± 0.01 | –0.27 ± 0.02 | 2.19 ± 0.04 | –2.47 ± 0.05 | 0.11 ± 0.02 |
| V58T | 19.06 ± 0.72 | –0.76 ± 0.02 | 0.061 ± 0.007 | 0.49 ± 0.01 | –0.71 ± 0.03 | 1.07 ± 0.07 | –1.79 ± 0.07 | 0.40 ± 0.04 |
The experimental conditions and analysis are described in Supporting Text. Errors correspond to fitting errors. 1 kcal = 4.18 kJ.
Refolding and unfolding rate constant in water
Dependence of the natural unfolding-refolding logarithm on urea
Gibbs free energy of refolding and unfolding of the mutants respect to D48G Gibbs free energy
Φ values for refolding
Value determined by A. R. Viguera, personal communication, and fixed for the fitting of the data by using Eq. 1 (Supporting Text). This mutant is the reference, because all Val mutations have been performed on it to counteract the expected large destabilization of the Thr and Ala mutations. D48G stabilizes the WT protein by 1.4 kcal·mol-1 and is located at the tip of a loop, thus not interfering with the mutations done here