Unbinding forces of single antibody-antigen complexes correlate with theirthermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructe d to obtain nine different single-chain Fv fragments, whose on-rates, off-r ates, and equilibrium binding affinities were determined in solution. Addit ionally, activation energies for unbinding were estimated from the temperat ure dependence of the off-rate in solution. Loading rate-dependent unbindin g forces were determined for single molecules by atomic force microscopy, w hich extrapolated at zero force to a value close to the off-rate measured i n solution, without any indication for multiple transition states. The meas ured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indi cating that the same transition state must be crossed in spontaneous and fo rced unbinding and that the unbinding path under load cannot be too differe nt from the one at zero force. The distance of the transition state from th e ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most l ikely reflects the elasticity of the protein in the unbinding process. Atom ic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level , predicting relative off-rates, potentially of great value for combinatori al chemistry and biology.