Table 1.

Thermodynamic parameters^a for urea-induced unfolding of variants and wild-type αTS at pH 7.8 and 25 °C^b

Secondary Structure	Variants	Kinetic Response	Equilibrium Unfolding						Refolding Burst Phase
			N⇌I1 transition			I1⇌ I2/U transition			ΔGo (H2O)	− m	Cm
			ΔGo (H2O)	−m	Cm	ΔGo (H2O)	− m	Cm
Cluster 2	WT		6.13 ± 0.09	2.29 ± 0.04	2.68 ± 0.06	4.72 ± 0.1	1.20 ± 0.04	3.93 ± 0.20	3.75 ± 0.25	1.08 ± 0.04	3.47 ± 0.26
β1	V23A	Group I	5.62 ± 0.06	2.58 ± 0.02	2.18 ± 0.03	2.22 ± 0.20	0.66 ± 0.04	3.36 ± 0.36	2.84 ± 0.80	0.64 ± 0.20	4.43 ± 1.86
	L25A		3.36 ± 0.59	2.16 ± 0.31	1.56 ±	1.67± 0.50	0.64 ± 0.11	2.61 ± 0.90	1.63 ± 0.18	0.55 ± 0.03	2.96 ± 0.36
α1	I37Ac		4.72 ± 0.14	2.72 ± 0.08	1.74 ± 0.07	1.71 ± 0.25	0.64 ± 0.06	2.67 ± 0.46	1.63 ± 0.73	0.55 ± 0.16	2.96 ± 1.58
	I41A		4.304.30	2.28 ± 0.07	1.89 ± 0.08	3.88 ± 0.51	1.09 ± 0.12	3.56 ± 0.61	3.57 ± 0.96	1.03 ± 0.21	3.47 ± 1.17
β2	L48A		1.27 ± 0.09	1.81 ± 0.07	0.70 ± 0.06	1.65 ± 0.04	0.64 ± 0.01	2.58 ± 0.07	1.76 ± 0.31	0.59 ± 0.04	2.98 ± 0.56
	L50Ac		5.99 ± 0.07	2.87 ± 0.04	2.09 ± 0.04	1.87 ± 0.11	0.70 ± 0.02	2.67 ± 0.17	2.06 ± 0.26	0.71 ± 0.05	2.90 ± 0.41
α2	L85A		5.00 ± 0.11	2.33 ± 0.05	2.15 ± 0.07	4.05 ± 0.62	1.23 ± 0.14	3.29 ± 0.63	3.92 ± 0.50	1.34 ± 0.15	2.92 ± 0.49
β3	I95A		4.80 ± 0.07	2.59 ± 0.04	1.85 ± 0.04	3.03 ± 0.18	0.97 ± 0.04	3.12 ± 0.23	3.40 ± 0.78	1.05 ± 0.21	3.24 ± 0.98
	I97A		3.08 ± 0.32	2.38 ± 0.23	1.29 ± 0.18	1.58 ± 0.28	0.63 ± 0.07	2.51 ± 0.53	1.75 ± 0.48	0.77 ± 0.12	2.27 ± 0.71
	L99Ac		4.44 ± 0.30	2.39 ± 0.18	1.86 ± 0.19	2.34 ± 0.51	0.94 ± 0.11	2.49 ± 0.62	2.34 ± 0.21	0.90 ± 0.04	2.60 ± 0.26
α3	L105A	Group II	5.07 ± 0.24	1.91 ± 0.10	2.65 ± 0.19	3.85 ± 0.58	1.03 ± 0.11	3.74 ± 0.69	2.81 ± 0.60	0.79 ± 0.11	3.56 ± 0.91
	I111A		3.76 ± 0.24	2.20 ± 0.14	1.71 ± 0.15	3.30 ± 0.25	0.94 ± 0.06	3.51 ± 0.35	2.35 ± 0.32	0.72 ± 0.05	3.26 ± 0.50
β4	V126A		5.21 ± 0.13	3.03 ± 0.08	1.72 ± 0.06	2.40 ± 0.08	0.69 ± 0.02	3.48 ± 0.15	1.66 ± 0.44	0.60 ± 0.05	2.77 ± 0.77
α8	L255A		3.77 ± 0.14	2.91 ± 0.10	1.30 ± 0.07	2.66 ± 0.08	0.75 ± 0.02	3.55 ± 0.14	1.84 ± 0.31	0.53 ± 0.03	3.47 ± 0.62
Cluster 1
α0	L11A		2.63 ± 0.21	2.02 ± 0.14	1.30 ± 0.14	3.55 ± 0.16	1.02 ± 0.04	3.48 ± 0.21	2.18 ± 0.80	0.71 ± 0.20	3.07 ± 1.42
β4	L127Ac		5.40 ± 0.08	2.55 ± 0.04	2.12 ± 0.05	4.17 ± 0.09	1.10 ± 0.02	3.79 ± 0.11	2.24 ± 0.20	0.71 ± 0.03	3.15 ± 0.31
β5	I151A		7.29 ± 0.10	2.66 ± 0.04	2.74 ± 0.06	4.32 ± 0.21	1.13 ± 0.04	3.82 ± 0.23	2.78 ± 0.24	0.82 ± 0.04	3.39 ± 0.34
β7	L209A		2.16 ± 0.15	2.36 ± 0.14	0.92 ± 0.08	3.51 ± 0.09	0.95 ± 0.02	3.69 ± 0.12	2.45 ± 0.26	0.73 ± 0.04	3.36 ± 0.40
β8	I232A		2.46 ± 0.21	1.49 ± 0.12	1.65 ± 0.19	4.31 ± 0.23	1.15 ± 0.05	3.75 ± 0.26	3.11 ± 0.48	0.94 ± 0.09	3.31 ± 0.60
Cluster 3
β6	L176Ac		3.25 ± 0.09	2.16 ± 0.05	1.50 ± 0.05	3.59 ± 0.07	1.01 ± 0.02	3.55 ± 0.10	2.82 ± 0.25	0.80 ± 0.04	3.53 ± 0.40

^aThe equilibrium unfolding data were fit to a 3-state model, N⇌I1⇌I2/U, while the refolding burst phase data were fit to a 2-state model, N⇌U, assuming a linear dependence of the free energy of unfolding on the denaturant concentration, ΔG^o=ΔG^o (H₂O)+m[urea].⁴¹ ΔGo , ΔG^o (H₂O), m and C_m represent the energy of unfolding in the presence of urea, the free energy of unfolding in the absence of urea, the urea dependence of the free energy of unfolding and the urea concentration at the midpoint of transition, respectively. Units are as follows: ΔG^o(H₂O), kcal mol⁻¹ (M urea)⁻¹; Cm, M urea. Errors for ΔG^o(H2O) and m are standard errors from the fits. Errors in C_m are obtained by standard error propagation of the equation: C_m=(H₂O)/m.

^bConditions: 10 mM potassium phosphate, pH 7.8, 0.2 mM K₂EDTA, and 1 mM β-mercaptoethanol.

^cData from ref.32