. 1998 Jul 21;95(15):8473–8478. doi: 10.1073/pnas.95.15.8473

Table 2.

Kinetics of refolding and unfolding of wild-type and mutant CI2

CI2 protein	k_f^{H₂^O}^*, s⁻¹	m_{k_f}^†, M⁻¹	ΔΔG_‡-D^{H₂^O}^‡ kcal mol⁻¹	k_u^{H₂^O}^*, ×10⁴ s⁻¹	m_{k_u}^†, M⁻¹	ΔΔG_‡-N^{H_2^O}^‡ kcal mol⁻¹
Wild type	56.5 ± 2.5	−1.82 ± 0.12	—	1.2 ± 0.1	1.31 ± 0.01	—
Mutations at the C terminus of the helix
KR18	66.0 ± 2.4	−1.91 ± 0.05	−0.09 ± 0.03	1.2 ± 0.2	1.26 ± 0.03	−0.01 ± 0.11
KR18/DR23	89.8 ± 3.6	−2.10 ± 0.06	−0.27 ± 0.04	28 ± 3	1.27 ± 0.02	−1.87 ± 0.01
DA23	83.9 ± 1.7	−2.16 ± 0.06	−0.23 ± 0.03	3.8 ± 0.9	1.38 ± 0.04	−0.69 ± 0.05
Mutations in the protease-binding loop region
RF48	2300 ± 200	−1.89 ± 0.04	−2.18 ± 0.06	n.d.	n.d.	−0.75 ± 0.02^§
RF48/FL50	375 ± 10	−1.92 ± 0.03	−1.12 ± 0.03	18 ± 3	1.14 ± 0.03	−1.59 ± 0.10

The rates of folding, k_f^{H_2^O}, and unfolding, k_u^{H_2^O}, of the protein were measured as described (6). The values of k_f^{H_2^O} measured directly in water (data not shown) and those derived by extrapolation from GdmCl (shown here) are identical within experimental error.

^†

m_{k_f} and m_{k_u} are calculated from the denaturant dependency of the folding and unfolding rate constants according to: ln k_f^[GdmCl] = ln k_f^{H_2^O} + m_{k_f} [GdmCl] and ln k_u^[GdmCl] = ln k_u^{H_2^O + m_{k_u} [GdmCl].}

^‡

ΔΔG_‡-D^{H_2^O} and ΔΔG_‡-N^{H_2^O} are the changes in free energies of activation of folding and unfolding, respectively, relative to the wild-type protein. They are calculated according to ref. 6.

^§

Owing to the high stability of the RF48 CI2 mutant, unfolding measurements were performed in the presence of Gdm SCN instead of GdmCl. The rate of unfolding of CI2 shows a large curvature in Gdm SCN, so the unfolding rates cannot be extrapolated to water accurately. The rates of unfolding in GdmSCN were fitted to the equation:

The kinetic parameters were calculated at 4 M Gdm SCN. Wild-type CI2 unfolds with a rate constant of 31.5 ± 1 s⁻¹ in 4 M Gdm SCN relative to 113 ± 3 s⁻¹ for RF48 CI2. We can thus calculate ΔΔG_‡-D^{4M Gdm SCN} for the RF48 CI2 mutant to be −0.75 ± 0.02 kcal mol⁻¹. The unfolding m_{k_u}_{[Gdm SCN]}-values in Gdm SCN are 1.69 ± 0.03 M⁻¹ and 1.58 ± 0.04 M⁻¹, for wild-type CI2 and RF48 CI2 respectively.

GdmCl, guanidine hydrochloride; Gdm SCN, guanidinium isothiocyanate.