Table 1. Thermodynamic and kinetic parameters of folding of thioredoxin variants 25 °C and pH 7.0.

Thermodynamic parameters of GdmCl-dependent unfolding/refolding equilibria of the thioredoxin variants, and folding rates Trx WTox and Trx1Pox involving the trans-to-cis isomerization of Pro76*
Trx variant			meq (kJ mol−1 M−1)		D1/2 (M GdmCl)		ΔD1/2a (M GdmCl)		ΔΔG0ox/red at the mean D1/2b (kJ mol−1)		kItrans→Ncis (s−1)c
Trx WTox			15.9 ± 0.34		2.24		0.74		12.0 ± 0.3		3.11 ± 0.20 · 10−3
Trx WTred			16.5 ± 0.50		1.50						n.d.g
Trx0Pox			9.53 ± 0.43		1.97		−0.79		−7.6 ± 0.4		n.a.h
Trx0Pred			9.82 ± 0.64		2.76						n.a.h
Trx1Pox			13.3 ± 0.29		2.85		0.06		0.8 ± 0.1		1.16 ± 0.15 · 10−4
Trx1Pred			11.8 ± 0.32		2.79						n.d.g
Kinetic parameters of unfolding and refolding that are independent of proline cis/trans isomerization **
Trx variant	kFH2O (s−1)		kUH2O (s−1)		mFd (M−1)		m U d (M) −1		ΔG0kin (kJ mol−1)		mkin (kJ mol−1 M−1)
Trx0Pox	7.70 ± 0.76		7.00 ± 1.3 ∙ 10−3		−1.69 ± 0.10		2.17 ± 0.06		−17.4 ± 2.0		9.56 ± 0.40e
Trx1Pox	n.d.g		9.56 ± 0.12 ∙ 10−7		n.d.g		3.55 ± 0.05		n.d.g		n.d.g
Trx1Pox Itrans	7.52 ± 0.77f		1.14 ± 0.62 ∙ 10−4 f		n.a.h		n.a.h		−27.5 ± 2.4f		12.7 ± 1.89f

^aDifference between the transition midpoints of both redox forms, defined as D_{1/2 (ox)}−D_{1/2 (red)}.

^bTo avoid errors due to extrapolation to zero denaturant, the differences between the free energies of folding of the oxidized and reduced forms was calculated at the GdmCl concentrations corresponding to the mean value of the respective transition midpoints (D_{1/2 mean}). D_{1/2 mean} values were 1.87, 2.37 and 2.82 M GdmCl for Trx WT, Trx0P and Trx1P, respectively. The ΔΔG⁰_{ox/red values} are defined such that positive values mean that the oxidized form is more stable than the reduced form.

^cRate constant of the rate-limiting step in the folding of the majority of molecules (94% in the case of Trx WT_ox and 95% in the case of Trx1P_ox) involving the trans-to-cis isomerization of the Ile75-Pro76 peptide bond, determined in the presence of 0.2 M GdmCl by interrupted refolding experiments (cf. Fig. 4a and Supplementary Fig. 3).

^dKinetic m values, corresponding to the linear dependence of ln k_F and ln k_U on GdmCl concentration.

^eEquilibrium m value predicted from kinetic data with the equation m_kin = (m_U–m_F)·RT.

^fThe GdmCl dependence of the observed rate of folding/unfolding of I_trans (Fig. 4c) was evaluated according to a mechanism with a high-energy on-pathway intermediate (U↔I↔N) with k_F = k_UI and k_U = k_NI(k_IU/k_IN)³³): m_kin = (Σ|m_i|) · RT, with m_UI = −0.58 M⁻¹, m_IN = −2.22 M⁻¹, m_NI = 2.33 M⁻¹ (m_IU was fixed to zero); k_UI = 7.52 s⁻¹, k_IN = 1.44 · 10⁵ s⁻¹, and k_NI = 1.64 · 10⁻³ s⁻¹ (k_IU was fixed to 10⁴ s⁻¹). The deduced transition midpoint of I_trans is 2.17 M GdmCl.

^gnot determined.

^hnot applicable.

^*Indicated errors correspond to errors from the fits described in the legends of Figs 3a and 4a.

^**Indicated errors correspond to errors from the fits decribed in the legends to Fig. 4a,c.