Table 1.
Parameters of the Pressure-Induced Transition in Single-Cysteine CYP3A4 Mutants Labeled with MDCC Observed by Pressure-Perturbation Fluorescence Spectroscopy
| Ligand | Position of the Label | P1/2 (Bar) | ΔV (mL/mol)a | ΔG (kJ/mol) | K°eqb |
|---|---|---|---|---|---|
| None | C85 | 1163 ± 315 | −29.4 ± 3.8 (<0.01)∗ | −3.42 ± 1.02 | 0.26 ± 0.12 |
| C409 | 617 ± 144 | −32.0 ± 4.6 (<0.01)∗ | −2.02 ± 0.83 | 0.46 ± 0.16 | |
| C64 | 134 ± 731 | −13.1 ± 4.4 (0.16) | −0.26 ± 1.01 | 0.92 ± 0.37 | |
| C166 | −802 ± 447 | −11.8 ± 3.2 (0.23) | 0.90 ± 0.27 | 1.44 ± 0.15 | |
| C468 | −73 ± 1327 | −11.5 ± 0.6 (0.19) | 0.10 ± 1.53 | 1.10 ± 0.65 | |
| C495 | 878 ± 873 | −13.7 ± 0.6 (0.06) | −1.22 ± 1.25 | 0.63 ± 0.31 | |
| BCT, 15 μM | C85 | 1066 ± 789 | −31.0 ± 14.0 (0.03)∗ | −3.01 ± 0.95 | 0.30 ± 0.11 (0.69) |
| C409 | 935 ± 855 | −30.3 ± 10.2 (<0.01)∗ | −3.18 ± 3.72 | 0.43 ± 0.44 (0.80) | |
| C64 | −344 ± 404 | −11.2 ± 4.4 (0.36) | 0.34 ± 0.30 | 1.15 ± 0.14 (0.38) | |
| C166 | −1399 ± 1875 | −11.3 ± 2.0 (0.24) | 1.48 ± 1.84 | 1.95 ± 1.39 (0.34) | |
| C468 | 202 ± 369 | −12.7 ± 0.2 (0.09) | −0.26 ± 0.47 | 0.91 ± 0.17 (0.64) | |
| C495 | 103 ± 1672 | −13.6 ± 2.5 (0.08) | −0.25 ± 2.29 | 1.01 ± 0.87 (0.51) | |
| TST, 200 μM | C85 | 179 ± 81 | −10.2 ± 5.6 (0.61) | −0.19 ± 0.18 | 0.93 ± 0.07 (<0.01)∗ |
| C409 | −713 ± 291 | −15.6 ± 2.3 (0.01)∗ | 1.20 ± 0.68 | 1.65 ± 0.47 (0.01)∗ | |
| C64 | −525 ± 323 | −12.4 ± 2.8 (0.15) | 0.67 ± 0.55 | 1.32 ± 0.29 (0.24) | |
| C166 | −488 ± 434 | −11.0 ± 0.8 (0.26) | 0.53 ± 0.44 | 1.24 ± 0.22 (0.69) | |
| C468 | −1009 ± 1395 | −10.8 ± 0.1 (0.28) | 1.09 ± 1.50 | 1.63 ± 0.95 (0.46) | |
| C495 | 412 ± 620 | −11.5 ± 1.9 (0.22) | −0.50 ± 0.79 | 0.83 ± 0.26 (0.45) | |
| None | GSH | 1245 ± 258 | −9.2 ± 3.9 | −1.17 ± 0.72 | 0.63 ± 0.18 |
The values given in the table represent the averages of the results of three to six individual experiments. The individual estimates of ΔV° and P1/2 were obtained from fitting of the pressure dependencies of the loading factor of the first PC found with PCA with Eq. 4. These estimated were used to calculate the values of ΔG and K°eq given in the table. The “±” values show the confidence interval calculated for p = 0.05.
Values in parentheses represent the p-values from Student’s t-test for the hypothesis of equality of the respective values of ΔV° with that determined for MDCC-GSH adduct in solution; ∗p ≤ 0.05 emphasizes the cases with a statistically significant difference from the case of protein-free label in solution.
Values in parentheses represent the p-values from Student’s t-test for the hypothesis of equality of the values of K°eq determined in the presence of BCT or TST with those observed in the absence of ligand; ∗∗p ≤ 0.05 emphasizes the cases with a statistically significant effect of ligands on the position of equilibrium.