KINETIC EPITOPE MAPPING OF THE CHICKEN LYSOZYME-CENTER-DOT-HYHEL-10 FAB COMPLEX - DELINEATION OF DOCKING TRAJECTORIES

Citation
Mg. Taylor et al., KINETIC EPITOPE MAPPING OF THE CHICKEN LYSOZYME-CENTER-DOT-HYHEL-10 FAB COMPLEX - DELINEATION OF DOCKING TRAJECTORIES, Protein science, 7(9), 1998, pp. 1857-1867
Citations number
54
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0961-8368
Volume
7
Issue
9
Year of publication
1998
Pages
1857 - 1867
Database
ISI
SICI code
0961-8368(1998)7:9<1857:KEMOTC>2.0.ZU;2-G
Abstract
The rate constants, k(on), for the formation of hen (chicken) lysozyme (HEWL).Fab-10 complexes have been determined for wild-type (WT) and e pitope-mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10-fold range [(0.18 to 1.92) X 10(6) M-1 s(-1)]. The affini ty constants, K-D, cover a broader, 440-fold, range from 0.075 to 33 n M. Values of K-D as high as 7 mu M were obtained for the complexes pre pared from some mutations at HEWL positions 96 and 97, but the associa ted kinetic constants could not be determined. The values of k(on) are negatively correlated with side-chain volume at position 101(HEWL), b ut are essentially independent of this parameter for position 21(HEWL) substitutions. The multiple mutations made at positions 21(HEWL) and 101(HEWL) provide sufficient experimental data on complex formation to evaluate Phi values [Phi = (Delta Delta G(on)(double dagger))/(Delta Delta G(D))] at these two positions to begin to define trajectories fo r protein-protein association. The data, when interpreted within the c oncept of a two-step association sequence embracing a metastable encou nter complex intermediate, argue that the rate determining step at pos ition 21(HEWL) (Phi(avg) = 0.2) is encounter complex formation, but th e larger Phi(avg) value of 0.36 experienced for most position 101(HEWL ) mutations indicates a larger contribution from the post-encounter an nealing process at this site for these replacements.