Table 1.

Binding kinetics and prediction method for measured A6 TCR point mutants.

Mutation1	Pred.2	ka, ×104 M−1s-1	kd, ×10−1 s−1	KD_wt/KD_mut3	ΔΔG, kcal/mol4
WT		5.11	1.08	1.0	0.00
alpha chain
D26M	R	-	-	0.7	0.19 ± 0.11
D26V	Z	-	-	0.1	1.45 ± 0.17
D26W	Z	2.44	0.08	6.2	−1.08 ± 0.13
R27F	Z	8.03	1.18	1.4	−0.22 ± 0.13
G28A	I	3.72	2.10	0.4	0.58 ± 0.13
G28I	I	4.27	0.58	1.6	−0.27 ± 0.11
G28L	I	6.92	0.68	2.2	−0.46
G28M	R	8.59	0.77	2.4	−0.51 ± 0.21
G28R	R	-	-	0.02	2.43 ± 0.49
G28T	Z	6.76	0.39	3.6	−0.76 ± 0.12
G28V	I	2.48	1.34	0.4	0.56 ± 0.16
S29A	I	3.91	1.45	0.6	0.33 ± 0.11
Q30N	I	2.88	2.09	0.3	0.73 ± 0.17
Q30E	Z	-	-	0.5	0.43 ± 0.14
S51M	Z	5.38	0.61	1.9	−0.37 ± 0.22
K68H	Z	-	-	0.2	1.09
S100A	I	4.71	0.97	1.0	−0.01 ± 0.14
S100N	I	-	-	0.04	1.92 ± 0.84
S100T	I	2.58	0.24	2.3	−0.49 ± 0.10
S100Y	I	-	-	0.02	2.34 ± 0.55
beta chain
I54R	R	-	-	0.1	1.28 ± 0.84
A99M	L	3.63	0.45	1.7	−0.31 ± 0.11
A99K	I	2.08	0.52	0.9	0.10 ± 0.14
G100S	L	2.48	0.31	1.7	−0.32 ± 0.10
G101A	L	1.86	0.06	6.3	−1.09 ± 0.10
R102Q	L	2.27	1.03	0.5	0.45 ± 0.23

¹The point mutation tested. WT = wild-type A6 TCR. Mutations in bold are those from ZAFFI predictions which improved binding.

²The prediction algorithm responsible for the mutation. I = our initial method; R = Rosetta (using Rosetta score); Z = ZAFFI; L = from Li et al. quadruple mutant {Li, 2005 #147}

³Binding affinity improvement, calculated as K_D of the wild-type A6 TCR (measured value = 2.11 μM) divided by K_D of the specified mutant.

⁴Binding energy change from the wild-type, with standard deviation calculated by separate analysis of three concentration gradients. For G28L and K68H, the uncertainty could not be calculated because only two gradients were used.

“−” kinetics not measurable, K_D obtained by steady-state analysis of the sensorgram. Otherwise K_D obtained by k_off/k_on