Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c.
Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.
Paterson
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003574:Cytochrome c Group / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D006860:Hydrogen Bonding; D007202:Indicators and Reagents; D008954:Models, Biological; D008958:Models, Molecular; D010446:Peptide Fragments / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D051381:Rats
D000906:Antibodies; D003574:Cytochrome c Group; D000939:Epitopes; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides