Table 1.

Thermodynamic and kinetic parameters of I_NC

Method	Conditions	ΔGIU, kcal/mol	kUI, sec−1	kIU, sec−1	kIN, sec−1
H/D pulse labeling (27)	pH 6.2, 10°C, 0.7 M GdmCl	3.0	55	0.3	2.7
N hydrogen-exchange (9)	pD 7.0, 30°C, 0.7 M GdmCl	2.3	61	1.3	10.0
Linear extrapolation	pH 6.2, 10°C, 1.0 M GdmCl	2.5	50	0.6	3.5

ΔG_IU values, obtained from hydrogen-exchange pulse labeling (27) or by the N hydrogen-exchange method (9) were taken from the literature where indicated or were computed from equilibrium unfolding data and the extrapolation of kinetic folding data as in Fig. 4. Values for k_UI were measured directly from the early phase of kinetic stopped-flow experiments done under three-state folding conditions, as in Fig. 2A. From these parameters, k_IU was evaluated by using ΔG_IU = −RT ln (k_IU/k_UI) and then compared with k_IN, measured from the later phase of the same stopped-flow experiments as for k_UI. These multiple comparisons all show that k_IN > k_IU, ruling out the off-pathway option, I ↔ U ↔ N.