Polypeptide-antibody binding mechanism: conformational adaptation investigated by equilibrium and kinetic analysis.
The mechanism of polypeptide-antibody binding was analysed by kinetic and equilibrium studies to find out whether or not the binding of an antibody to a large protein or a polypeptide antigen behaves as a one-step reaction or involves conformational adaptation. Three monoclonal antibodies recognizing 3 distinct epitopes on the C-terminal domain (F2) of Escherichia coli tryptophan synthase beta 2 subunit were used. The dissociation equilibrium constant (KD), the association rate constant (kon) and the dissociation rate constant (koff) of these antibodies for the native beta 2 subunit, its C-terminal fragment (F2) and different polypeptides obtained by chemical cleavage of the F2 fragment were measured. It was found that for some polypeptide-antibody complexes, binding could not be described as a one-step association-dissociation reaction, thus indicating the existence of conformational adaptation upon antibody-antigen complex formation. It was also shown that differences in affinities of a given antibody for its epitope carried by different polypeptides were mainly due to differences in the dissociation rate constant (koff) and not in the association rate constant (kon). Moreover, the immunoreactivity of various polypeptides obtained by cleavage of the F2 fragment enabled us to localize the 3 epitopes on the beta chain in light of the 3-dimensional structure of tryptophan synthase described recently by Hyde et al. (1988).
Friguet
B
B
Unité de Biochimie Cellulaire et Moléculaire, Institut Pasteur, Paris.
Djavadi-Ohaniance
L
L
Goldberg
M E
ME
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D014367:Tryptophan Synthase / Q000235:genetics*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D010455:Peptides; D014367:Tryptophan Synthase