Table 1. Thermodynamic stability, folding speed, and φ values for WT and point-mutated apo-azurin variants (pH 7, 25°C).
Apo-azurin variant | Mutated β-strand | ΔGU(H2O), kJ/mol | ΔΔGU(H2O), kJ/mol | kF(H2O), s-1 | ΔΔG‡(H2O), kJ/mol | φ† |
---|---|---|---|---|---|---|
WT | — | 25.0 ± 0.7 | — | 160 ± 20 | — | — |
Val-31—Ala | 3 | 20.0 ± 0.4 | -5.0 ± 0.7 | 23 ± 3 | -4.72 ± 0.3 | 0.93 ± 0.14 |
Leu-33—Ala | 3 | 21.0 ± 0.3 | -4.0 ± 0.7 | 35 ± 2 | -3.70 ± 0.3 | 0.91 ± 0.18 |
Trp-48—Ala | 4 | 4.0 ± 0.3 | -21.0 ± 0.7 | 22 ± 6 | -4.83 ± 0.7 | 0.23 ± 0.03§ |
Leu-50—Ala | 4 | 18.0 ± 0.2 | -7.0 ± 0.7 | 5 ± 1 | -7.3 ± 0.3 | 1.04 ± 0.11 |
Val-95—Ala | 6 | 22.5 ± 0.2 | -2.5 ± 0.7 | 78 ± 3 | -1.75 ± 0.3 | 0.63 ± 0.21¶ |
Phe-97—Ala | 6 | 15.0 ± 0.7 | -10.0 ± 0.7 | 40 ± 6 | -3.38 ± 0.3 | 0.35 ± 0.04 |
Tyr-108—Ala | 7 | 12.5 ± 0.2 | -12.5 ± 0.7 | 23 ± 3 | -4.72 ± 0.3 | 0.37 ± 0.03 |
Phe-110—Ala | 7 | 11.0 ± 0.9 | -14.0 ± 0.9 | 54 ± 5 | -2.64 ± 0.3 | 0.19 ± 0.02 |
The φ values are calculated as φ = ΔΔG‡(H2O)/ΔΔGU(H2O), where ΔΔGU(H2O) is calculated as ΔGU(mutant,H2O)-ΔGU(WT,H2O) and ΔΔG‡(H2O) as RTln[kf(mutant,H2O)/kf(WT,H2O)].
Standard deviations (σ) for the φ values were calculated as in ref. 19: σφ = |φ|*√[(σΔΔG‡/ΔΔG‡)2+(σΔΔGU/ΔΔGU))2].
Not used in analysis because this value may contain large errors; ΔGU(H2O) is only 4.0 kJ/mol and the refolding rate constant in water had to be estimated, assuming a two-state reaction, using the equilibrium stability and the unfolding speed.
Not used in analysis because ΔGU(mutant,H2O)-ΔGU(WT,H2O) is only 2.5 kJ/mol and the φ-value estimation may be unreliable.