Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein alpha-helix.
Monoclonal antibodies against an alpha-helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D003198:Computer Simulation; D000939:Epitopes; D006422:Hemerythrin / Q000031:analogs & derivatives / Q000276:immunology; D008667:Metalloproteins / Q000276:immunology; D008958:Models, Molecular; D009038:Motion; D010455:Peptides / Q000276:immunology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006422:Hemerythrin; D008667:Metalloproteins; D010455:Peptides; C008571:myohemerythrin