A. Rajpal et al., QUANTITATIVE-EVALUATION OF THE CHICKEN LYSOZYME EPITOPE IN THE HYHEL-10 FAB COMPLEX - FREE-ENERGIES AND KINETICS, Protein science, 7(9), 1998, pp. 1868-1874
The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal
antibody HyHEL-10 Fab (Fab-10) was investigated by alanine scan mutag
enesis. The association rate constants (k(on)) for the HEWL.Fab-10 com
plexes were obtained from the homogenous solution method described in
the preceding paper (Taylor et al., 1998), A new method for determinin
g the dissociation rate constant (k(off)) for the complex, by trapping
nascent free antibody with an inactive HEWL mutant is described. The
values of k(on) fall within a factor of 2 of the wild-type (WT) HEWL v
alue (1.43 +/- 0.13 x 10(6) M-1 s(-1)), while the increases in k(off)
more nearly reflect the total change in free energies of the complex (
Delta Delta G(D)). The dissociation constants (K-D) were measured dire
ctly in those cases where satisfactory kinetic data could not be obtai
ned. The Y20A, K96A, and K97A HEWL.Fab-10 complexes are destabilized b
y more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and
D101A antibody complexes are moderately destabilized (0.7 < Delta Del
ta G(D) less than or equal to 1.0 kcal/mol). Additional mutations of t
he ''hotspot'' residues (Tyr20, Lys96, Lys97) were constructed to prob
e, more precisely, the nature of their contributions to complex format
ion. The results show that the entire hydrocarbon side chains of Tyr20
and Lys97, and only the E-amino group of Lys96, contribute to the sta
bility of the complex. The value of Delta Delta G(D) for the R21A muta
nt complex is a distinct outlier in the Arg21 replacement series demon
strating the importance of supplementing alanine scan mutagenesis with
additional mutations.