Table 2.

Equilibrium and kinetic parameters for divalent metal ion binding¹

Site	Mutation (location)	ΔΔGmut	$\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{equil}}(\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{kin}})$ 2	$\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{f}}^{‡}(\mathrm{\Delta }{\text{G}}_{\text{f}}^{‡})$	m0 ( ${\text{m}}_0^{\text{Me}}$)	mf/m0 ( ${\text{m}}_{\text{f}}^{\text{Me}}/{\text{m}}_0^{\text{Me}}$)	${\psi }_0^{\text{Leffler}}({\psi }_0^{\text{chevron}})$ 3	Metal
WT	NA	NA	NA	NA (1.51 ± 0.06)	1.97 ± 0.08 (NA)	0.73 ± 0.05 (NA)	NA	NA
a	I11H T17H (β1-β2)	0.14 ± 0.02	0.88 ± 0.034 (0.70 ± 0.18)	0.46 ± 0.17 (0.79 ± 0.05)	2.11 ± 0.11 (1.92 ± 0.06)	0.67 ± 0.05 (0.63 ± 0.03)	0.77 ± 0.07 (0.51 ± 0.17)	Zn
b	N9H T19H (β1–β2)	−0.32 ± 0.02	1.31 ± 0.13 (1.11 ±0.24)	0.77 ± 0.22 (1.64 ± 0.16)	2.38 ± 0.22 (1.68 ± 0.12)	0.76 ± 0.11 (0.67 ± 0.08)	0.75 ± 0.03 (0.48 ± 0.07)	Zn
c	I11H K61H (β1-β4)	−0.45 ± 0.03	ND (0.92 ± 0.10)	1.03± 0.10 (1.18 ± 0.06)	1.81 ± 0.07 (1.58 ± 0.06)	0.65 ± 0.04 (0.68 ± 0.04)	1.24 ± 0.07 (1.28 ± 0.10)	Zn
d	N9H N59H (β1-β4)	0.53 ± 0.02	1.03 ± 0.15 (0.85 ± 0.16)	1.63 ± 0.25 (1.88 ± 0.05)	1.87 ± 0.11 (1.70 ± 0.07)	0.73 ± 0.06 (0.69 ± 0.04)	3.33 ± 0.40 (4.64 ± 1.24)	Zn
e	A52H T57H (β3-β4)	−0.88 ± 0.02	−0.60 ± 0.075 (−0.60 ± 0.03)	−0.76 ± 0.05 (1.11 ± 0.02)	2.22 ± 0.07 (2.38 ± 0.11)	0.81 ± 0.04 (0.82 ± 0.06)	1.13 ± 0.03 (1.13 ± 0.02) 6	Zn
f	D50H N59H (β3-β4)	0.64 ± 0.02	0.21 ± 0.17 (0.03 ± 0.12)	0.66 ± 2.47 (1.79 ± 0.10)	1.96 ± 0.13 (1.54 ± 0.11)	0.73 ± 0.08 (0.65 ± 0.08)	ND 7	Zn
g	K28H E32H (helix)	−0.46 ± 0.02	1.10 ± 0.04 (0.76 ± 0.24)	0.29 ± 0.16 (1.37 ± 0.05)	1.92 ± 0.14 (1.81 ± 0.11)	0.69 ± 0.08 (0.66 ± 0.06)	0.26 ± 0.03 (0.25 ± 0.09)	Zn
			1.20 ± 0.12 (0.92 ± 0.22)	−0.13 ± 0.16 (1.37 ± 0.05)	1.92 ± 0.14 (1.57 ± 0.09)	0.69 ± 0.08 (0.61 ± 0.06)	NA 8 (−0.05 ± 0.05)	Ni
h	A35H T39H (helix)	0.30 ± 0.01	0.48 ± 0.11 (0.33 ± 0.20)	−0.38 ± 0.65 (1.32 ± 0.05)	2.14 ± 0.12 (1.78 ± 0.13)	0.71 ± 0.06 (0.73 ± 0.08)	ND 7	Zn
			1.75 ± 0.07 (2.01 ± 0.16)	0.28 ± 0.08 (1.32 ± 0.05)	2.14 ± 0.12 (1.98 ± 0.06)	0.71 ± 0.06 (0.75 ± 0.04)	NA 8 (0.02 ± 0.004)	Ni

¹Units are kcal · mol⁻¹ (free energies) or kcal · mol⁻¹ ·M⁻¹ (m values). NA, not applicable. ND, not determined.

² $\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{equil}}$ is the metal-induced stabilization determined by 280nm fluorescence versus GdmCl concentration curve from equilibrium denaturation measurement unless mentioned otherwise. $\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{kin}}$ is the metal-induced stabilization obtained from the simultaneous fit of double chevrons. To minimize extrapolation errors, changes in stabilities were calculated at 0.5 and 4.5M GdmCl, and are obtained by simultaneously fitting two chevrons in the absence and presence of 1 mM metal ion, with the parameter of interest being one of the fitting parameters.

³ ${\psi }_0^{\text{Leffler}}$ and ${\psi }_0^{\text{chevron}}$ are the ψ₀values obtained from double chevron analysis and the fit of a Leffler plot, respectively.

⁴ $\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{equil}}$ is determined by $\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{equil}}=\mathrm{\Delta }{C}_m·{\text{m}}_0^{\text{No}\text{metal}}$ where ΔC_m is the metal-induced change in mid-point, which is the GdmCl concentration where the folded and unfolded populations are the same. ${\text{m}}_0^{\text{No}\text{metal}}$ is the m₀ obtained in the absence of metal ion.

⁵ $\mathrm{\Delta }\mathrm{\Delta }{\text{G}}_{\text{eq}}^{\text{equil}}$ is determined by equilibrium denaturation using circular dichroism measurements at λ = 222nm instead of florescence measurement. The m₀ value was shared when equilibrium denaturation data in the absence and presence of metal ion were fitted simultaneously.

⁶The slope of unfolding arm m_u was shared when two chevrons were simultaneously fitted.

⁷Chevron shifts vertically upon addition of metal ion. The metal-induced stabilization is too small to provide a well-defined value.

⁸Unfolding arm has two phases of which the fast and slow phase exchange their relative amplitudes as the nickel ion concentration increases while the rates themselves are constant over nickel ion concentration. The ψ₀ value could not be obtained from the Leffler plot because ΔΔG_eq cannot be determined at the intermediate nickel ion concentration where the relative amplitude of the two phases is poorly determined.