Figure 9. Comparison of the impact of single wild-type reversion mutations on the binding affinity and thermodynamic stability of the P4 V_H domain.

The affinity (K_A) and stability (T_m^*) measurements for the P4 variants containing single wild-type reversion mutations were evaluated as described in Figs 2 and 3, respectively. Decreases in V_H affinity or stability due to reversion mutations signify that the P4 mutations contribute positively to affinity or stability in the corresponding V_H domain (and vice versa for increases in V_H affinity or stability due to reversion mutations). The reversion mutations are highlighted in grey (framework residue), red (CDR2), blue (CDR3) and green (CDR4). The K_A measurements for the P4 mutants are averages of four to five repeats, while the T_m^* measurements are averages of two repeats (the error bars are standard deviations). The solid and dotted lines are the averages and standard deviations (respectively) for the original P4 V_H domain (seven repeats for the association constant, two repeats for the apparent melting temperature).