Table 2.

Calculated and experimental values of the changes of free energy, ΔΔG, of the interaction between IFN-γ-Rx mutants and IFN-γ-SC relative to the wild-type receptor.

Construct		The best ΔΔG c	ΔΔG from MDd	Experimental ΔΔG e	esdf
IDa	Mutationb	(kJ/mol)	(kJ/mol)	(kJ/mol)	(kJ/mol)
1	N65R	−5.4	17.3	2.1	—
2	N70G	−5.4	0.3	−0.6	—
3	S95R	−8.3	11.8	2.1	—
4	N96F	−13.0	−0.6	−0.2	—
5	N96W	−9.9	−6.1	−3.9	0.2
6	K115Y	−0.3	−9.6	0.7	—
7	T166M	−5.8	−5.4	2.0	—
8	T166Y	−9.8	0.9	2.5	—
9	H222R	−6.9	−15.8	−0.1	0.2
10	N96W + H222R	−7.1	−7.1	−5.0	0.2
11	N70G + S95R	−7.3	2.7	1.5	—
12	N70G + H222R	−4.6	−7.3	−0.3	—
13	S95R + H222R	−11.4	−10.8	1.5	—
14	N70G + S95R + H222R	−15.8	−5.6	0.5	0.1
15	Y66L	2.1	11.8	0.0	—
16	S71E	9.6	19.6	1.6	—
17	H222D	6.7	5.8	2.0	—

^aMutants 1–14 are single, double, and triple mutants designed to increase affinity to IFN-γ compared to WT. Mutants 15–17 were designed to lower the affinity between IFN-γ and IFN-γ-Rx but not to destabilize the unbound IFN-γ-Rx.

^bResidues are numbered as in the UniProt entry P15260.

^cFor mutants 1–14, the most negative (most stabilizing) values obtained at the four crystal interfaces by FoldX [33]. For mutants 15–17, the ΔΔG listed are for the least positive (least destabilizing) interface.

^dAveraged ΔΔG values calculated by FoldX on structures taken from snapshots of 10 to 20 ns MD runs by GROMACS [36].

^eΔΔG values determined from experimental SPR values of dissociation equilibrium constants K _d as ΔΔG = −RTln⁡{(K _d)_WT/(K _d)_mut}.

^fEstimated standard deviations for the experimental values of ΔΔG with the number of independent SPR measurements N > 2 (Table 3).