Table 1.

Thermodynamic and kinetic parameters of CRABP1 variants^a

Variant	Locationb	ΔΔGU-N (kcal/mol)c	ln kUd	mU (kcal/mol·M)d	ΔΔG‡-N (kcal/mol)e	φ-valuef
wild typeg		n/a	−11.0 ± 0.1	0.64 ± 0.02	n/a	n/a
F3I+ h	Strand 1	−3.6 ± 0.2	−6.5 ± 0.1	0.80 ± 0.03	−2.6 ± 0.1	0.26 ± 0.05
W7Y	Strand 1	−2.2 ± 0.2	−9.7 ± 0.2	0.68 ± 0.02	−0.8 ± 0.1	0.65 ± 0.07
M9A+	Strand 1′	−1.8 ± 0.2	−11.5 ± 0.2	0.87 ± 0.02	0.3 ± 0.2	1.17 ± 0.09
S12G+	Strand 1′	−1.8 ± 0.2	−10.38 ± 0.08	0.706 ± 0.008	−0.34 ± 0.08	0.82 ± 0.05
F15A	Helix I	−2.9 ± 0.2	−8.5 ± 0.2	0.65 ± 0.01	−1.25 ± 0.08	0.57 ± 0.04
L18A+	Helix I	−3.5 ± 0.2	−9.5 ± 0.4	0.88 ± 0.05	−0.9 ± 0.2	0.75 ± 0.07
L19A− i	Helix I	−3.3 ± 0.2	−7.58 ± 0.07	0.547 ± 0.005	−2.00 ± 0.09	0.39 ± 0.05
L22A+	Helix I	−2.7 ± 0.2	−10.43 ± 0.02	0.859 ± 0.002	−0.31 ± 0.08	0.88 ± 0.03
R29A−	Helix II	−0.8 ± 0.3	−9.17 ± 0.04	0.458 ± 0.003	−1.06 ± 0.08	n/a
V31A	Helix II	−0.6 ± 0.3	−10.6 ± 0.2	0.587 ± 0.008	−0.2 ± 0.1	n/a
V33A−	Helix II	−0.4 ± 0.3	−9.9 ± 0.5	0.54 ± 0.05	−0.6 ± 0.3	n/a
I43V	Strand 2	−1.6 ± 0.2	−8.9 ± 0.1	0.61 ± 0.05	−1.2 ± 0.1	0.3 ± 0.1
F50I	Strand 3	−1.7 ± 0.2	−8.51 ± 0.04	0.66 ± 0.01	−1.45 ± 0.08	0.1 ± 0.1
V58A	Turn II	−0.2 ± 0.2	−10.9 ± 0.2	0.642 ± 0.002	0.0 ± 0.1	n/a
K66A−	Turn III	−1.8 ± 0.3	−7.85 ± 0.07	0.48 ± 0.01	−1.84 ± 0.09	0.0 ± 0.2
V67A	Turn III	−3.3 ± 0.2	−6.5 ± 0.2	0.70 ± 0.03	−2.7 ± 0.1	0.18 ± 0.06
G68A−	Turn III	−2.6 ± 0.2	−6.9 ± 0.2	0.50 ± 0.03	−2.4 ± 0.2	0.06 ± 0.09
E69A−	Turn III	−1.3 ± 0.2	−9.02 ± 0.07	0.51 ± 0.01	−1.15 ± 0.09	0.1 ± 0.1
G70A	Turn III	0.7 ± 0.2	−11.9 ± 0.1	0.69 ± 0.01	0.6 ± 0.1	0.2 ± 0.2
T75A−	Turn IV	−2.4 ± 0.3	−9.0 ± 0.1	0.54 ± 0.05	−1.2 ± 0.1	0.51 ± 0.08
V76A	Turn IV	−1.5 ± 0.3	−10.3 ± 0.1	0.675 ± 0.007	−0.4 ± 0.1	0.74 ± 0.08
D77A−	Turn IV	−1.0 ± 0.3	−9.56 ± 0.07	0.529 ± 0.001	−0.83 ± 0.09	0.2 ± 0.2
G78A−	Turn IV	−2.5 ± 0.4	−9.53 ± 0.04	0.547 ± 0.009	−0.85 ± 0.08	0.66 ± 0.06
R79A−	Turn IV	−3.1 ± 0.3	−8.6 ± 0.2	0.46 ± 0.02	−1.4 ± 0.1	0.55 ± 0.06
W87F	Strand 6	−2.6 ± 0.4	−7.7 ± 0.1	0.59 ± 0.02	−1.9 ± 0.1	0.2 ± 0.1
I93V−	Strand 7	−1.8 ± 0.2	−8.49 ± 0.05	0.567 ± 0.004	−1.46 ± 0.08	0.2 ± 0.1
W109I−	Strand 8	−2.9 ± 0.2	−7.6 ± 0.1	0.37 ± 0.02	−2.0 ± 0.1	0.31 ± 0.07
L113V	Strand 8	−0.8 ± 0.2	−10.48 ± 0.02	0.66 ± 0.01	−0.28 ± 0.08	n/a
L118V+	Strand 9	−2.3 ± 0.4	−9.9 ± 0.2	0.79 ± 0.03	−0.7 ± 0.2	0.72 ± 0.08
C129A−	Strand 10	−0.6 ± 0.2	−9.8 ± 0.1	0.55 ± 0.01	−0.68 ± 0.09	n/a
Q131A−	Strand 10	−2.0 ± 0.2	−8.15 ± 0.02	0.516 ± 0.006	−1.66 ± 0.08	0.17 ± 0.09
Y133S+	Strand 10	−4.8 ± 0.2	−9.32 ± 0.02	0.950 ± 0.001	−0.97 ± 0.08	0.80 ± 0.02
R135G+	Strand 10	−3.9 ± 0.3	−8.7 ± 0.1	0.71 ± 0.01	−1.34 ± 0.09	0.66 ± 0.04

^aThe average values from at least two independent measurements and the standard deviation are reported; all values reported are extrapolated to 0 M denaturant

^bSecondary structural location of the selected variant residue

^cThe energetic effect of mutations on the free energy of the unfolded state (U) with respect to the native state (N) calculated as ΔΔG_U-N = ΔG_U-N^WT−ΔG_U-N^mut where ΔG_U-N^WT was determined to be 10.0 ± 0.2 kcal/mol, and ΔG_U-N values were obtained by fitting equilibrium urea unfolding curves using m_eq = −2.0 kcal/mol·M (Fig. 2A,C)

^dObtained from linear extrapolation of ln k_U versus [urea] (Fig. 2B,D)

^eThe energetic effect of mutation on the free energy of the transition state (‡) with respect to the native state (N), calculated as ΔΔG_‡-N = −RT ln (k_U^WT/k_U^mut)

^fCalculated as φ_F = 1 − φ_U

^gHis-tagged CRABP1 with a stabilizing R131Q mutation was used as WT

^hDesignates variants with m_U > 0.7 kcal/mol·M (m_U+)

ⁱDesignates variants with m_U < 0.57 kcal/mol·M (m_U−)