Mechanical Strength of Specific Bonds Acting Isolated or in Pairs: A Case Study on Engineered Proteins

The dynamic strength of multiple specific bonds exposed to external mechanical force is of significant interest for the understanding of biological adhesion. Exploiting the well-established FLAG tag technology, we engineered model proteins exhibiting no, one, or two identical binding sites for a monoclonal antibody. Bonds between these engineered proteins and the antibody were studied with dynamic force spectroscopy. On single bonds between a FLAG-tag and the antibody, we observed two regimes corresponding to two different activated complexes, that is, two intermediate states along the reaction path for bond breakage. Dynamic force spectroscopy on double bonds showed the same two regimes. The actual yield forces of double bonds slightly exceeded those of single bonds. A simplified kinetic model with analytical solutions was developed and used to interpret the measured spectra.