Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

The hen (chicken) egg‐white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL‐I0 Fab (Fab‐I0) was investigated by alanine scan mutagenesis. The association rate constants (kon) for the HEWL.Fab‐10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of kon fall within a factor of 2 of the wild‐type (WT) HEWL value (1.43 ± 0.13 × l06 M−1 s−1), while the increases in koff more nearly reflect the total change in free energies of the complex (ΔΔGD). The dissociation constants (KD) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL Fab‐I0 complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and Dl0lA antibody complexes are moderately destabilized (0.7 > ΔΔGD < 1.0 kcal/mol). Additional mutations of the “hotspot” residues (TYRO, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the ϵ‐amino group of Lys96, contribute to the stability of the complex. The value of ΔΔGD for the R21A mutant complex is a distinct outlier in the Arg21 replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.