Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c.
Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.
Collawn
J F
JF
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Wallace
C J
CJ
Proudfoot
A E
AE
Paterson
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000138:chemical synthesis / Q000235:genetics* / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D005818:Genetic Engineering; D006736:Horses; D008958:Models, Molecular; D011487:Protein Conformation; D011863:Radioimmunoassay / Q000379:methods
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003574:Cytochrome c Group; D000939:Epitopes