Table 1.
Parameters determined from the best fit of the folding/unfolding kinetics of Im7 solvation variants
| Variant | Position | KUI (MUI) | kIN (mIN) | kNI (mNI) | ΔG°UI (kJ mol− 1) | ΔG°UN (kJ mol− 1) | ΦI | ΦTS2 | βI | βTS2 |
|---|---|---|---|---|---|---|---|---|---|---|
| IV7Ta | N term | kUN 279.4 ± 10.69 (mUN 3.27 ± 0.06) | kNU 3.40 ± 0.14 (mNU 0.68 ± 0.01) | − 1.84 ± 1.22a | − 10.38 ± 0.13 | 0.73b | 1.00 ± 0.04 | 0.83 ± 0.005 | ||
| F15Y | Helix I | 13.9 ± 3.02 (4.25 ± 0.18) | 220.2 ± 14.77 (0.64 ± 0.34) | 5.95 ± 0.12 (0.48 ± 0.01) | − 6.19 ± 0.51 | − 14.69 ± 0.54 | 0.62 ± 0.03 | 0.65 ± 0.03 | 0.79 ± 0.05 | 0.91 ± 0.02 |
| V16T | Helix I | 52.9 ± 7.30 (4.18 ± 0.06) | 244.5 ± 9.57 (0.49 ± 0.11 | 1.79 ± 0.08 (0.55 ± 0.02) | − 9.34 ± 0.32 | − 20.90 ± 0.35 | 0.79 ± 0.07 | 0.81 ± 0.06 | 0.80 ± 0.02 | 0.90 ± 0.01 |
| V27T | Loop 1 | 137.7 ± 16.49 (4.17 ± 0.05) | 250.2 ± 8.60 (0.37 ± 0.07) | 0.69 ± 0.08 (0.51 ± 0.04) | − 11.59 ± 0.28 | − 25.46 ± 0.39 | ND | ND | 0.83 ± 0.01 | 0.90 ± 0.01 |
| V36T | Helix II | 77.6 ± 11.29 (3.99 ± 0.06) | 276.6 ± 11.63 (0.32 ± 0.1) | 1.05 ± 0.08 (0.68 ± 0.03) | − 10.24 ± 0.34 | − 23.35 ± 0.40 | ND | ND | 0.80 ± 0.02 | 0.86 ± 0.01 |
| F41Y | Helix II | 80.0 ± 46.95 (3.37 ± 0.46) | 186.1 ± 15.74 (0.84 ± 0.17) | 40.43 ± 9.83 (0.40 ± 0.07) | − 10.31 ± 1.38 | − 13.90 ± 1.51 | 0.21 ± 0.10 | 0.28 ± 0.01 | 0.73 ± 0.04 | 0.91 ± 0.03 |
| V42T | Helix II | 300.2 ± 62.79 (4.27 ± 0.10) | 125.0 ± 3.30 (0.47 ± 0.05) | 3.19 ± 0.19 (0.41 ± 0.02) | − 13.42 ± 0.49 | − 22.06 ± 0.52 | − 0.23 ± 0.30 | 0.31 ± 0.17 | 0.83 ± 0.01 | 0.92 ± 0.01 |
| IV54Ta | Helix III | 53.43 ± 6.21 (4.06 ± 0.08) | 229.5 ± 11.90 (0.03 ± 0.13) | 0.66 ± 0.07 (0.62 ± 0.04) | − 9.36 ± 0.27 | − 23.13 ± 0.40 | − 0.89 ± 0.60 | − 0.95 ± 0.38 | 0.86 ± 0.02 | 0.87 ± 0.01 |
| Y55F | Helix III | 133.4 ± 22.41 (3.89 ± 0.06) | 286.7 ± 9.77 (0.58 ± 0.07) | 1.28 ± 0.12 (0.55 ± 0.03) | − 11.51 ± 0.40 | − 24.25 ± 0.46 | ND | ND | 0.78 ± 0.01 | 0.89 ± 0.01 |
| V69T | Helix IV | 20.01 ± 2.08 (4.51 ± 0.23) | 225.8 ± 21.52 (0.42 ± 0.29) | 0.53 ± 0.05 (0.68 ± 0.03) | − 7.05 ± 0.24 | − 21.30 ± 0.40 | 1.45 ± 0.17 | 1.53 ± 0.16 | 0.80 ± 0.04 | 0.88 ± 0.01 |
| IV72Ta | Helix IV | 110.9 ± 13.70 (4.00 ± 0.05) | 273.7 ± 9.66 (0.48 ± 0.08) | 0.79 ± 0.07 (0.47 ± 0.03) | − 11.08 ± 0.29 | − 24.84 ± 0.36 | ND | ND | 0.85 ± 0.04 | 0.88 ± 0.01 |
| F84Y | C term | 7.55 ± 2.04 (3.25 ± 0.18) | 114.4 ± 5.64 (1.02 ± 0.35) | 1.01 ± 0.02 (0.80 ± 0.01) | − 4.76 ± 0.64 | − 15.88 ± 0.65 | 0.90 ± 0.03 | 1.06 ± 0.03 | 0.64 ± 0.05 | 0.84 ± 0.03 |
| Im7 | 222.2 ± 55.56 (4.19 ± 0.10) | 253.9 ± 11.46 (0.75 ± 0.09) | 1.26 ± 0.14 (0.45 ± 0.04) | − 12.71 ± 0.59 | − 25.19 ± 0.65 | — | — | 0.78 ± 0.01 | 0.92 ± 0.01 |
Φ-values were calculated from kIU, kIN and kNI. For IV7T, the data were fitted to equations describing a two-state model of folding. βT values, which describe the solvent exposure of I or TS relative to the native state, were calculated from kinetic m values according to the equation:
The units of k are s− 1; units of M/m are kJ mol− 1 M− 1.
Indicates proteins for which Φ-values have been calculated using a pseudo wild-type (the corresponding I→V variant).
The chevron plot for this variant was fitted to equations describing apparent two-state folding and the maximum possible ΔG°UI estimated by modelling the minimum ΔG°UI required for refolding branch rollover to be observed (∼ 1 kJ mol− 1). The difference between the ΔG°UN obtained for IV7T from equilibrium denaturation (ΔG°UN − 12.22 ± 1.21 kJ mol− 1, data not shown) and kinetic experiments (ΔG°UN − 10.38 ± 0.13 kJ mol− 1) provides another means of estimating ΔG°UI. With this approach, a ΔG°UI of − 1.84 ± 1.22 kJ mol− 1 was determined for the IV7T variant. ND Φ-values were not calculated for these variants as ΔΔG°UN was too small (< 2.5 kJ mol− 1).34,35