Table 3.
Kinetic and thermodynamic parameters for the folding/unfolding kinetics of Im7H3M3 variants
| Protein | Location in native structure | KUI (MUI) | KIN (mIN) | KNI (mNI) | ΔGUIo | ΔGUNo |
|---|---|---|---|---|---|---|
| Im7H3M3 | — | 544.5 ± 92.9 (4.7 ± 0.2) | 158.3 ± 4.1 (0.4 ± 0.1) | 4.26 ± 0.3 (− 0.7 ± 0.0) | − 14.8 ± 0.4 | − 23.3 ± 0.4 |
| A13G | Helix I (exposed) | 155.4 ± 31.4 (4.7 ± 0.2) | 174.6 ± 12.9 (0.5 ± 0.2) | 2.4 ± 0.14 (− 0.9 ± 0.0) | − 11.9 ± 0.5 | − 21.9 ± 0.5 |
| V16A | Helix I (buried) | 29.2 ± 5.2 (4.7 ± 0.2) | 216 ± 8.8 (0.3 ± 0.1) | 7.1 ± 0.19 (− 0.7 ± 0.0) | − 7.9 ± 0.4 | − 15.9 ± 0.4 |
| L19A | Helix I (buried) | 24.7 ± 3.0 (4.7 ± 0.2) | 259 ± 56.13 (1.8 ± 1.3) | 24.5 ± 0.51 (− 1 ± 0.0) | − 7.5 ± 0.9 | − 13.1 ± 0.9 |
| K24A | Helix I (exposed) | 333.3 ± 96.3 (4.7 ± 0.2) | 142.3 ± 6.7 (0.2 ± 0.1) | 5.99 ± 0.56 (− 0.7 ± 0.0) | − 13.7 ± 0.7 | − 21.1 ± 0.7 |
| A24G | Helix I (exposed) | 368.1 ± 37.0 (4.7 ± 0.2) | 157.1 ± 6.3 (0.4 ± 0.1) | 7.89 ± 0.27 (− 0.7 ± 0.0) | − 13.9 ± 0.2 | − 20.9 ± 0.3 |
| V33A | Helix II (exposed) | 874.6 ± 68.8 (4.7 ± 0.2) | 143.8 ± 3.9 (0.6 ± 0.0) | 4.36 ± 0.1 (− 0.9 ± 0.0) | − 15.9 ± 0.2 | − 24.1 ± 0.2 |
| D35A | Helix II (exposed) | 3092.8 ± 223.2 (4.7 ± 0.2) | 85.2 ± 2.19 (0.3 ± 0.03) | 4.19 ± 0.28 (− 0.7 ± 0.0) | − 18.9 ± 0.2 | − 25.9 ± 0.2 |
| A35G | Helix II (exposed) | 1818.2 ± 165.3 (4.7 ± 0.2) | 99.6 ± 4.6 (0.17 ± 0.06) | 7.5 ± 0.58 (− 0.6 ± 0.0) | − 17.6 ± 0.2 | − 23.7 ± 0.3 |
| L37A | Helix II (buried) | 35.7 ± 10.5 (4.7 ± 0.2) | 382.0 ± 27.4 (0.7 ± 0.3) | 9.7 ± 0.4 (− 1.5 ± 0.0) | − 8.4 ± 0.7 | − 17.0 ± 0.7 |
| E39A | Helix II (exposed) | 1734 ± 210 (4.7 ± 0.2) | 134.7 ± 7.4 (0.55 ± 0.08) | 8.79 ± 0.68 (− 0.5 ± 0.0) | − 17.5 ± 0.3 | − 24.0 ± 0.4 |
| A39G | Helix II (exposed) | 1851 ± 171 (4.7 ± 0.2) | 63.0 ± 2.3 (0.49 ± 0.05) | 20.4 ± 0.66 (− 0.4 ± 0.0) | − 17.7 ± 0.2 | − 20.4 ± 0.3 |
| F41L | Helix II (buried) | 170.5 ± 32.0 (4.7 ± 0.2) | 421.7 ± 26.7 (0.1 ± 0.1) | 10.3 ± 0.55 (− 0.7 ± 0.0) | − 12.1 ± 0.4 | − 20.8 ± 0.5 |
| V42A | Helix II (partially buried) | 57.8 ± 17.8 (4.7 ± 0.2) | 585.3 ± 41.8 (0.8 ± 0.2) | 6.45 ± 0.68 (− 0.9 ± 0.0) | − 9.5 ± 0.7 | − 20.1 ± 0.8 |
| T51S | Helix III (buried) | 290.1 ± 28.1 (4.7 ± 0.2) | 204.2 ± 9.0 (0.5 ± 0.0) | 4.62 ± 0.24 (− 1 ± 0.0) | − 13.4 ± 0.2 | − 22.3 ± 0.3 |
| A52G | Helix III (exposed) | 288.5 ± 66.6 (4.7 ± 0.2) | 177.5 ± 2.9 (0.5 ± 0.0) | 5.3 ± 0.3 (− 0.9 ± 0.0) | − 13.3 ± 0.6 | − 21.6 ± 0.6 |
| L53A | Helix III (buried)a | 163.9 ± 59.1 (4.7 ± 0.2) | − | − | − 17.4 ± 2.0 | − |
| I54V | Helix III (buried) | 222.2 ± 37.9 (4.7 ± 0.2) | 225.7 ± 14.1 (0.5 ± 0.1) | 4.80 ± 0.19 (− 1.3 ± 0.0) | − 12.7 ± 0.4 | − 21.8 ± 0.4 |
| AIIG | Helix III (exposed) | 238.1 ± 30.2 (4.7 ± 0.2) | 355 ± 15.1 (1.2 ± 0.1) | 11.6 ± 0.28 (− 1 ± 0.0) | − 12.9 ± 0.3 | − 20.9 ± 0.3 |
| AVIG | Helix III (exposed) | 137.0 ± 16.3 (4.7 ± 0.2) | 221.4 ± 9.5 (0.3 ± 0.1) | 5.9 ± 0.18 (− 0.7 ± 0.0) | − 11.6 ± 0.3 | − 20.1 ± 0.3 |
| V69A | Helix IV (buried) | 219.0 ± 16.0 (4.7 ± 0.2) | 192.6 ± 6.6 (0.3 ± 0.1) | 5.2 ± 0.17 (− 0.9 ± 0.0) | − 12.7 ± 0.2 | − 21.2 ± 0.2 |
| K70A | Helix IV (exposed) | 576.9 ± 36.6 (4.7 ± 0.2) | 148.9 ± 4.6 (0.2 ± 0.05) | 6.7 ± 0.3 (− 0.6 ± 0.0) | − 14.9 ± 0.2 | − 22.3 ± 0.2 |
| A70G | Helix IV (exposed) | 87.5 ± 12.7 (4.7 ± 0.2) | 240.3 ± 11.9 (0.7 ± 0.1) | 6.0 ± 0.18 (− 0.8 ± 0.0) | − 10.5 ± 0.3 | − 19.2 ± 0.4 |
| A77G | Helix IV (exposed) | 140.8 ± 23.1 (4.7 ± 0.2) | 132. ± 7.2 (0.9 ± 0.1) | 8.8 ± 0.28 (− 0.6 ± 0.0) | − 11.6 ± 0.4 | − 18.0 ± 0.4 |
| A78G | Helix IV (exposed) | 91.7 ± 14.6 (4.7 ± 0.2) | 123.3 ± 6.2 (0.4 ± 0.1) | 9.2 ± 0.2 (− 0.7 ± 0.0) | − 10.6 ± 0.4 | − 16.7 ± 0.4 |
All rate constants are expressed in s− 1, all m-values are expressed in kJ mol− 1 M− 1 and all free energies are expressed in kJ mol− 1. Data were acquired at 10 °C in 50 mM sodium phosphate (pH 7.0) and 0.4 M Na2SO4. Errors were calculated as described in Friel et al.2
L53A folds predominantly (> 95%) to an intermediate state. The rates of folding of this variant versus the rates of folding of the denaturant were fitted to a two-state transition, yielding kUI (450.7 ± 25.3 s− 1) and kIU (10.3 ± 0.7 s− 1) from which ΔGUIo (and MUI) values were calculated. Equilibrium studies confirmed that these parameters equate to those determined kinetically (ΔGUIo=− 17.4 ± 2.0 kJ mol− 1 and MUI = 4.7 ± 0.5 kJ mol− 1 M− 1, determined by equilibrium denaturation monitored by far-UV CD at 222 nm).