Kinetic analysis of the effect on fab binding of identical substitutions in a peptide and its parent protein

Monoclonal antibody 57P, which was raised against tobacco mosaic virus prot ein, crossreacts with a peptide corresponding to residues 134-146 of this p rotein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics t he structure in the protein. In this study, we carried out a detailed compa rison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the pa rent protein, and the effect of these substitutions on antibody binding par ameters have been measured with a Biacore instrument. Fabs that recognize e pitopes located away from the site of mutations were used as indirect probe s for the conformational integrity of protein antigens. Their interaction k inetics with all proteins were similar, suggesting that the substitutions h ad no drastic effect on their conformation. The five substitutions introduc ed in the peptide and the protein had minor effects on association rate con stants (k(a)) and significant effects on dissociation rate constants (k(d)) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope wa s presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation, Howeve r, k(a) values were about 10 times larger and k(d) values about 5 times lar ger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed f ur three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and prote in antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to m imic correctly some but not all properties of the protein-Fab 57P interacti on and highlight the importance of quantitative analysis of both equilibriu m and kinetic binding parameters in the design of synthetic vaccines and dr ugs.