QUANTITATIVE-EVALUATION OF THE CHICKEN LYSOZYME EPITOPE IN THE HYHEL-10 FAB COMPLEX - FREE-ENERGIES AND KINETICS

Citation
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
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0961-8368
Volume
7
Issue
9
Year of publication
1998
Pages
1868 - 1874
Database
ISI
SICI code
0961-8368(1998)7:9<1868:QOTCLE>2.0.ZU;2-7
Abstract
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.