Table 2. Folding properties of IκBα mutants.
| A. Folding properties of IκBα67-206W mutants | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment: | Equilibriuma | Kineticsb | ||||||
| Mutant | CD ΔGeq | Fluorescence ΔGeq | k12 | k21 | k32 | k43 | ΔGeqc | Figure |
| IκBα67-206W | 6.5±0.2 | 6.9±0.1 | 2.4 | 2.4 × 104 | 57 | 0.71 | 5.7 | dashed line |
| S76T/F77P | 6.9±0.2 | 7.5±0.1 | 3.0 | 2.1 × 104 | 51 | 0.55 | 6.1 | 4a |
| V93L | 8.6±0.2 | 9.1±0.1 | 2.0 | 0.91 × 104 | 13 | 0.25 | 7.5 | 4b |
| Q111G | 8.4±0.2 | 9.2±0.1 | 3.7 | 2.0 × 104 | 4.7 | 0.30 | 7.9 | 4c |
| T113S | 5.6±0.2 | 6.1±0.1 | 2.1 | 1.3 × 104 | 68 | 1.3 | 5.5 | 4d |
| L117V | 5.5±0.2 | 5.8±0.1 | 2.2 | 1.4 × 104 | 89 | 1.7 | 5.2 | 4e |
| N122G | 7.5±0.2 | 8.2±0.1 | 2.7 | 1.5 × 104 | 20 | 0.78 | 6.6 | 4f |
| A127V | 6.8±0.2 | 7.3±0.1 | 1.8 | 0.79 × 104 | 12 | 1.5 | 6.6 | 4g |
| L131V | 4.7±0.2 | 5.0±0.1 | 2.6 | 2.0 × 104 | 186 | 1.1 | 4.9 | 4h |
| T146S | 4.9±0.2 | 5.3±0.1 | 2.3 | 1.6 × 104 | 123 | 0.93 | 5.3 | 4i |
| V160A | 6.7±0.2 | 7.2±0.1 | 2.7 | 1.8 × 104 | 39 | 0.75 | 6.1 | 4j |
| L163V | 5.2±0.2 | 5.6±0.1 | 0.65 | 1.7 × 104 | 114 | 0.55 | 4.9 | 4k |
| T164L | 7.3±0.2 | 7.8±0.1 | 9.7 | 0.76 × 104 | 75 | 0.61 | 7.1 | 4l |
| T185S | 5.7±0.2 | 6.1±0.1 | 4.0 | 4.3 × 104 | 42 | 1.0 | 5.6 | 4m |
| C186P | 7.2±0.2 | 7.7±0.1 | 2.1 | 1.6 × 104 | 28 | 0.67 | 6.3 | 4n |
| G194A | 5.4±0.2 | 5.8±0.1 | 1.0 | 3.1 × 104 | 38 | 0.98 | 5.1 | 4o |
| V203L | 7.3±0.2 | 7.8±0.1 | 4.0 | 0.50 × 104 | 33 | 0.72 | 7.2 | 4p |
| B. Folding properties of IκBα67-287W mutants | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment: | Equilibriuma | Kineticsb | ||||||
| Mutant | CD ΔGeq | Fluorescence ΔGeq | k12 | k21 | k32 | k43 | ΔGeqc | Figure |
| IκBα67-287W | 7.1±0.2 | 7.4±0.3 | 0.48 | 1.3 × 103 | 25 | 0.37 | 7.1 | 5b |
| W258F | 7.2±0.3 | 7.4±0.3 | 0.97 | 4.0 × 103 | 30 | 0.36 | 6.8 | 5a |
| Q111G | 8.7±0.3 | 9.2±0.3 | 0.55 | 0.94 × 103 | 2.4 | 0.18 | 9.1 | 6b |
| T164L | 7.5±0.3 | 7.5±0.3 | 1.8 | 0.47 × 103 | 139 | 28 | 7.6 | 6d |
| V203L | 7.4±0.3 | 7.4±0.3 | 0.70 | 0.88 × 103 | 38 | 0.35 | 7.3 | 6f |
Folding stabilities (ΔGeq) are reported in kcal mol-1 and rates in s-1.
Equilibrium denaturation experiments measured by circular dichroism or total fluorescence. All IκBα67-206W mutants were globally fit to a two-state model with baseline drift with shared m-values of 2.19 kcal mol-1 M-1 for CD and 2.35 kcal mol-1 M-1 for fluorescence. IκBα67-287W mutants were also globally fit with shared m-values of 2.13 kcal mol-1 M-1 for CD and 2.20 kcal mol-1 M-1 for fluorescence. Reported errors are fit errors from the global fit of all mutants.
Kinetics experiments were measured by stop-flow fluorescence. All IκBα67-206W mutants were globally fit to a four-state model with shared m-values (in kcal mol-1 M-1): m12 = -0.11; m21 = 0.98; m32 = 0.89; m43 = 0.11; IκBα67-287W mutants were similarly globally fit to a four-state model with shared m-values (in kcal mol-1 M-1): m12 = -0.15; m21 = 0.96; m32 = 0.87; m43 = 0.11. For both, m23 and m34 were set to 0 kcal mol-1 M-1 and k23 and k34 were set to 1 × 105 s-1. The equilibrium m-value calculated from the m-values for all rate constants is meq = -m12 + m21 + m32 + m43 = 2.10 kcal mol-1 M-1 for IκBα67-206W mutants and 2.09 kcal mol-1 M-1 for IκBα67-287W mutants, both in agreement with the equilibrium measurement. To determine experimental error, three independent data sets for wild type IκBα67-206W at 10°C were globally fit with shared m-values. Individual rates were determined for each data set; from these, the standard deviation in each rate was calculated as 14% for k12, 22% for k21, 22% for k32, and 2.6% for k43.
ΔGeq was calculated from the individual rates: ΔGeq = R T ln (k43 * k32 * k21 / (k12 * k23 * k34)). Standard deviation in ΔGeq was calculated to be 5.7%, based on propagation of the standard deviations of the rates.