Table 1.

Fitted parameters for two-state equilibrium unfolding and three-state kinetic refolding and unfolding PP32 variants

Variant	$k_{\mathrm{D}\mathrm{I},{\text{H}}_2\text{O}}$	$k_{\mathrm{I}\mathrm{D},{\text{H}}_2\text{O}}$	$k_{\mathrm{I}\mathrm{N},{\text{H}}_2\text{O}}$	$k_{\mathrm{N}\mathrm{I},{\text{H}}_2\text{O}}$	$m_{\mathrm{D}\mathrm{I}}$	$m_{\mathrm{I}\mathrm{D}}$	$m_{\mathrm{I}\mathrm{N}}$	$m_{\mathrm{N}\mathrm{I}}$	$\mathrm{\Delta }{G}_{{\text{H}}_2\text{O},eq}^{\mathrm{o}}$	$\mathrm{\Delta }{G}_{{\text{H}}_2\text{O},kin}^{\mathrm{o}}$	$m_{eq}$	$m_{kin}$
Wild-type	5.60	0.53	3.47 × 105	2.40	−0.88	0.36*	−1.36	0.35*	7.93 ± 0.18	8.29 ± 0.01	2.86 ± 0.02	2.95 ± 0.02
I7A	6.38	0.80	3.08 × 105	36	−0.88	0.45*	−1.48	0.16*	5.86 ± 0.06	6.48 ± 1.87	2.85 ± 0.04	2.97 ± 0.04
L11A	6.93	2.01	3.83 × 104	4.78	−0.91	0.36†	−1.22	0.35†	5.92 ± 0.10	5.95 ± 0.04	2.95 ± 0.04	2.85 ± 0.02
V19A	6.55	1.55	2.53 × 105	4.57	−0.89	0.37*	−1.52	0.33*	6.69 ± 0.17	7.20 ± 0.06	3.05 ± 0.13	3.11 ± 0.02
L22A	6.99	2.52	6.66 × 102	2.51	−0.92	0.36†	−0.72	0.35†	3.88 ± 0.05	3.84 ± 0.01	2.36 ± 0.03	2.34 ± 0.02
L37A	6.52	2.76	5.35 × 102	2.23	−0.93	0.36†	−0.72	0.35†	3.68 ± 0.15	3.69 ± 0.08	2.28 ± 0.07	2.36 ± 0.11
L47A	7.51	3.27	5.52 × 102	1.68	−0.87	0.36†	−1.00	0.35†	2.98 ± 0.17	3.86 ± 0.06	2.22 ± 0.06	2.58 ± 0.13
L60A	5.79	0.49	6.61 × 102	0.88	−0.80	0.36†	−1.79	0.35†	4.06 ± 0.10	5.29 ± 0.01	2.61 ± 0.01	3.31 ± 0.01
L69A	6.28	0.79	3.20 × 103	0.29	−0.88	0.60*	−1.74	0.57*	4.99 ± 0.13	6.63 ± 0.01	3.02 ± 0.03	3.79 ± 0.02
L83A	9.40	0.97	8.40 × 101	0.89	−1.39	0.36†	−0.83	0.35†	4.46 ± 0.02	3.97 ± 0.01	3.25 ± 0.02	2.93 ± 0.04
L93A	5.26	0.88	6.07 × 102	0.98	−1.11	0.36†	−1.41	0.35†	4.95 ± 0.19	4.79 ± 0.04	3.47 ± 0.11	3.23 ± 0.18
L109A	0.89	0.65	5.23 × 102	0.57	−1.13	0.36†	−1.35	0.35†	4.56 ± 0.08	4.15 ± 0.01	3.37 ± 0.17	3.18 ± 0.02
L118A	0.71	1.07	3.13 × 102	1.14	−1.48	0.36†	−0.85	0.35†	3.60 ± 0.12	3.03 ± 0.03	3.26 ± 0.12	3.03 ± 0.03
Y131F	12.68	0.64	2.93 × 104	2.82	−1.02	0.36†	−1.30	0.35†	6.97 ± 0.09	7.13 ± 0.03	3.05 ± 0.04	3.03 ± 0.06
V135G	0.03	0.65	3.29 × 104	1.12	−1.21	0.36†	−1.46	0.35†	4.07 ± 0.19	4.13 ± 0.60	3.61 ± 0.16	3.38 ± 0.04
K137G	1.27	0.57	6.72 × 105	5.41	−0.92	0.36†	−1.39	0.35†	7.26 ± 0.12	7.30 ± 1.24	3.04 ± 0.17	3.02 ± 0.03
L139A	0.03	0.66	1.01 × 104	0.64	−0.77	0.36†	−1.45	0.35†	4.40 ± 0.16	3.85 ± 0.24	3.36 ± 0.11	2.93 ± 0.03
L142A	0.03	0.67	9.16 × 103	0.70	−1.03	0.36†	−1.41	0.35†	3.86 ± 0.19	3.77 ± 0.17	3.29 ± 0.15	3.15 ± 0.02
L145A	0.33	0.69	1.04 × 103	0.73	−1.33	0.36†	−1.22	0.35†	4.01 ± 0.18	3.79 ± 0.02	3.37 ± 0.09	3.26 ± 0.04
D146L	1.03	0.74	1.45 × 102	0.80	−1.30	0.36†	−0.84	0.35†	3.49 ± 0.26	3.22 ± 0.04	2.77 ± 0.22	2.85 ± 0.08
Y131F/D146L	4.53	0.60	1.41 × 103	109	−0.98	0.36†	−0.59	0.35†	4.72 ± 0.14	4.01 ± 0.09	2.69 ± 0.14	2.28 ± 0.14
ΔNCap PP32	6.93	2.57	4.36 × 103	2.31	−0.9	0.36†	−1.10	0.35†	4.13 ± 0.09	4.97 ± 0.10	2.37 ± 0.04	2.65 ± 0.12

Rate constants k in s⁻¹; $\Delta {\mathit{G}}_{{\mathrm{H}}_2\mathrm{O}}^{\mathrm{o}}$ in kcal⋅mol⁻¹; m-values in kcal⋅mol⁻¹⋅M⁻¹.

^*For these constructs, parameters were fitted simultaneously to major and minor observed rate constants and unfolding amplitudes.

^†For these variants, which show only one unfolding phase, m_ID and m_NI were fixed at the WT values. Errors for $\mathrm{\Delta }{G}_{{\text{H}}_2\text{O},eq}^{\mathrm{o}}$ and $m_{eq}$ are the SD of three independent experiments. Errors for $\mathrm{\Delta }{G}_{{\text{H}}_2\text{O},kin}^{\mathrm{o}}$ and $m_{kin}$ (m_DI – m_ID + m_IN – m_NI) are propagated from standard propagation formulas.