Table I.

Global Analysis of Equilibrium Denaturation Data of WT, P61A, and P61A/Y(38, 69, or 95)W FIS Mutants^a

Protein	Denaturant	ΔGN2 ⇆ I2 (kcal/mol)	mN2 ⇆ I2 [kcal/(mol M)]	ΔGI2 ⇆ 2U (kcal/mol)	mI2 ⇆ 2U [kcal/(mol M)]	ΔGN2 ⇆ 2U (kcal/mol)	mN2 ⇆ 2U [kcal/(mol M)]
WTb	GuHCl					13.1 ± 0.2	4.7 ± 0.2
WTb	Urea					14.7 ± 1.7	2.4 ± 0.1
P61Ac	GuHCl	4.9 ± 0.2	1.9 ± 0.08	11.5 ± 0.2	1.9 ± 0.07	16.4 ± 0.3	3.8 ± 0.1
P61Ab	Urea	6.1 ± 0.3	1.03 ± 0.05	12.5 ± 0.4	1.10 ± 0.06	18.6 ± 0.5	2.13 ± 0.08
P61A/Y38Wc	GuHCl	6.5 ± 0.1	2.5 ± 0.05	15.8 ± 0.2	2.9 ± 0.05	22.3 ± 0.2	5.4 ± 0.07
P61A/Y69W	GuHCl	4.4 ± 0.1	2.3 ± 0.06	13.3 ± 0.1	2.1 ± 0.03	17.7 ± 0.1	4.4 ± 0.07
P61A/Y95W	GuHCl	4.0 ± 0.1	1.5 ± 0.03	11.1 ± 0.1	1.7 ± 0.3	15.1 ± 0.1	3.2 ± 0.3

^aDenaturation curves of WT and P61A were fit to a N₂ ⇄ 2U denaturation mechanism. Denaturation curves of P61A and P61A/Y#W mutants were fit to a N₂ ⇄ I₂ ⇄ 2U denaturation mechanism. Most FIS variants were analyzed by CD and fluorescence spectroscopy, except P61A FIS, which was not amenable to fluorescence experiments due to its low Tyr fluorescence.

^bData and fitting of WT (GuHCl), WT (urea), and P61A (urea) denaturation are described in references31,28 and30, respectively.

^cGlobal analysis was performed on the P61A/Y#W mutants using Excel and parameter error was calculated using SOLVER-AID as described in Materials and Methods and references40 and41.