Figure 3
Comparison of SPR and SEC binding assays for evaluation of redesigned MICA proteins. Each MICA mutant from Table 1 is organized according to the results of the SEC binding assay and graphed against a different parameter. The average of each category is shown as a vertical line. (a) SEC results compared to RosettaDesign score [13] shows that negative RosettaDesign scores (predicted stabilized) only have binding results similar to or worse than wild-type, while positive RosettaDesign scores (predicted destabilized) include all 5 MICA mutants found to bind more tightly than wild-type. SEC results compared to SPR results [13] from (b) equilibrium and (c-d) kinetic analysis show similar correlations between equilibrium affinity and on-rate with the highest-affinity mutants by SEC, while no correlation with off-rate is seen because the set of mutants does not vary significantly in this parameter. Observed kon and koff rates from single-step kinetic fits for mutants predicted to be stabilized, and from two-step kinetic fits for mutants predicted to be destabilized (k+1 and k-2, respectively). (P values for the difference between ++ and + complexes: t-test P = 0.0013 for Figure 3a; 0.0006 for Figure 3b; 0.0001 for Figure 3c; and 0.6 for Figure 3d.)