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)/ΔΔG_U(H₂O), where ΔΔG_U(H₂O) is calculated as ΔG_U(mutant,H₂O)-ΔG_U(WT,H₂O) and ΔΔG^‡(H₂O) as RTln[k_f(mutant,H₂O)/k_f(WT,H₂O)].

^†Standard deviations (σ) for the φ values were calculated as in ref. 19: σ_φ = |φ|*√[(σ_ΔΔG‡/ΔΔG^‡)²+(σ_ΔΔGU/ΔΔG_U))²].

^§Not used in analysis because this value may contain large errors; ΔG_U(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 ΔG_U(mutant,H₂O)-ΔG_U(WT,H₂O) is only 2.5 kJ/mol and the φ-value estimation may be unreliable.