Fine manipulation of antibody affinity for synthetic epitopes by altering peptide structure: antibody binding to looped peptides*.

Linear peptides weakly imitate antibody binding sites on globular proteins when the peptides are shown to be effective at all. As a step toward enhancing the ability of peptides to mimic epitopes, we have examined the effects of various alterations in peptide structure on antibody binding. Synthetic peptides containing the core amino acid sequence of residues 41 to 48 from horse cytochrome c were examined for their ability to bind antibodies elicited against the 41-48 peptide coupled to bovine serum albumin (BSA). Since residues 41-48 in native cytochrome c are part of an omega loop, in some peptides cysteines were incorporated for intrachain disulfide bonding to stabilize loop structure. In additional cases, glycine was incorporated as a spacer between the natural sequence and the cysteine residues with the intent of relaxing loop structure slightly. Eleven analogues containing the 41-48 sequence were tested. These included native cytochrome c and the 1-80 and 1-65 cyanogen bromide-cleaved fragments. The native protein did not bind the anti-41-48 antibodies. The other analogues differed by over three orders of magnitude in their binding. The affinity of binding was inversely related to the extent of predicted loop structure indicating that the antibodies were elicited against the 41-48 sequence in a more unfolded conformation despite the Pro Gly sequence at positions 44 and 45 that generally favors a beta turn. Surprisingly, the immunizing peptide, containing residues 41-48 only, was the poorest binding peptide. The relative impotence of 41-48 was shown to be largely due to differences at the amino terminus between the free and BSA-coupled peptides as the antibodies were elicited against the latter. The distinctions among the synthetic peptides containing the 41-48 sequence show the exquisite sensitivity of antibody binding to amino acid changes that may occur outside of an epitope and suggest modifications in peptide structure at the periphery of an epitope that can lead to desired changes in antibody affinity.