Increasing Affinity of Interferon-𝜸 Receptor 1 to Interferon-𝜸 by Computer-Aided Design

Table 3

Affinity between IFN-𝛾-SC and IFN-𝛾-Rx mutants was predicted to increase affinity measured by surface plasmon resonance (SPR).

Construct		𝑘𝑎∗10^−6	𝑘𝑑∗10^2	𝐾𝑑	𝑁^e	esd (𝐾𝑑)
ID	Mutation^a	(1/Ms)^b	(1/s)^c	(nM)^d		(nM)^f
WT	—	1.24	3.78	30.8	14	1.5
1	N65R	0.882	6.28	71.2	1	na
2	N70G	1.12	2.64	23.6	1	na
3	S95R	0.650	4.54	69.8	2	na
4	N96F	1.01	2.83	28.0	1	na
5	N96W	1.43	0.909	6.34	4	0.49
6	K115Y	0.979	3.91	39.9	1	na
7	T166M	0.933	6.39	68.5	1	na
8	T166Y	0.940	7.82	83.1	1	na
9	H222R	1.19	3.49	29.4	6	1.9
10	N96W + H222R	2.40	1.00	4.16	3	0.37
11	N70G + S95R	0.889	4.94	55.9	2	na
12	N70G + H222R	1.46	3.91	26.9	2	na
13	S95R + H222R	1.05	5.90	56.3	2	na
14	N70G + S95R + H222R	1.09	4.01	37.0	5	2.1

aResidues are numbered as in UniProt P15260.
^bKinetic constant of association, 𝑘_𝑎.
^cKinetic constant of dissociation, 𝑘_𝑑.
^dDissociation equilibrium constants 𝐾_𝑑 calculated as 𝑘_𝑑/𝑘_𝑎.
^eNumber of independent SPR measurements.
^fValues of the estimated standard deviation (esd) of 𝐾_𝑑 are shown for mutants with three and more measurements (listed in column 𝑁).
Confidence limits calculated from the Students 𝑡-distribution at the 95% level are ±0.85, ±0.78, ±1.9, ±0.93, and ±2.6 nM for WT, N96W, H222R, N96W + H222R, and N70G + S95R + H222R, respectively.