Affinity measurements by sedimentation equilibrium and BIAcore, as well as the biological activity assays, were performed as described in Materials and Methods. Differences in free energy changes are calculated as the differences between ΔGs of mutant and wild-type SAGs (ΔΔG = ΔG_mutant − ΔG_wild-type). The values of the individual ΔGs are calculated from the average K_ds obtained from sedimentation equilibrium and BIAcore measurements according to the equation ΔG = −RT ln (1/K_d), where R is the universal gas constant and T is the absolute temperature in Kelvin. For the three weakest binding mutants, N23A, Y90A, and Q210, a ΔΔG value of >2.5 kcal/mol (which corresponds to a 70-fold decrease in affinity) is estimated, as these mutants bind at least 50–100-fold weaker than wild-type SEC3. The stimulatory capacity of different SAGs was estimated from the dose (ng/ml) required to induce proliferation 100-fold above background. Background cpm were typically less than 100. NB, no detectable binding observed in the BIAcore measurements at TCR-β chain concentrations up to 128 μM; ND, not determined.  

 150 mM should be considered as a lower limit for these mutants.  

 Value for SEB wild-type was taken from the earlier work by Malchiodi et al. (15).  

 For these SEC3 mutants, SAG concentrations of 30,000 ng/ml were not sufficient to induce proliferation 100-fold above background.