Table 2.

Stability of the proposed intermediate of wild-type barnase measured by different methods in kcal/mol at pH 6.3 and 25°C

	Direct methods			Indirect methods*
	Urea melting†	Competition HX‡	Native-state HX§
ΔGIU	−0.6	∼0.0	0.3	2.7 to 3.2

^*Values are 2.8 kcal/mol from Dalby et al. (24), 2.7 kcal/mol from the chevron plot fitting that used a three-state model with fixed intermediate and transition state (see Materials and Methods and Fig. 1), and 3.2 kcal/mol from Matouschek et al. (1). 

^† The value was obtained by fitting the CD signals (Fig. 3C) to a two-state model with floating parameters for both pretransition and posttransition baselines (Eq. 4). If K_IU is fixed with a value of 0.25 (ΔG_IU = 0.8 kcal/mol), the fitting yields an m_IU value that is 60% of the m_NU value for global unfolding (20). This value is significantly larger than the 43% value obtained from the chevron curve fitting (see Fig. 1), indicating that the ΔG_IU is unlikely larger than 0.8 kcal/mol. 

^‡ This value was obtained assuming an average protection factor of 2 (Fig. 2). Simulation studies that use Eq. 5 show that a ±10% error in P_occ(H) measurement may contribute an error less than a factor of 2 in P_f, which may contribute an uncertainty in ΔG_IU by ∼0.4 kcal/mol. 

^§ The value was calculated from ΔG_u^app – ΔG_HX (see text) based on the HX data of Clarke and Fersht (9). Possible uncertainties in k_int by a factor of 2 may contribute another error in free energy by 0.4 kcal/mol. Thus, the ΔG_IUs determined from both HX experiments are unlikely larger than 0.8 kcal/mol.