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

Abstract

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic 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 combinatorial chemistry and biology.

Binding Affinities of Host–Guest, Protein–Ligand, and Protein–Transition‐State Complexes

“…[30] Frequency plots similar to those for the systems described previously have been compiled based on a limited set of available data. [31] These antibody binding data were divided into two types: the binding constants for antibody complexes with organic molecules are shown in Figure 11, while those for antibody-protein and antibody-oligosaccharide complexes are shown in Figure 13. The repertoire of antibody types includes whole immunoglobulins (specifically, immunoglobulin G, IgG), Fab regions, and single-chain F V regions.…”

“…Plot of the number of antibody-antigen complexes found to fall within a particular range of Àlg K d (= lg K a ) values (K a = 10 7.3AE1.9 m À1 ). [31] The antigens here are relatively small organic molecules, and data for proteins and carbohydrates are excluded.…”

“…Plot of the number of antibody-antigen complexes of proteins and carbohydrates found to fall within a particular range of Àlg K d (= log K a ) values (K a = 10 8.9AE1.7 m À1 ). [31] Binding Affinities of Host-Guest Complexes Angewandte Chemie (haptens) that are analogues of transition states of organic reactions. Haptens are attached to proteins such as keyhole limpet hemocyanin or bovine serum albumin and then used to induce antibody production.…”

“…Plot of DH versus DS for antibody-antigen complexes; linear regression gives DH = 296.9(AE 28.2) DSÀ10.5(AE 0.8). [31] Figure 15. Plot of the number of complexes formed between catalytic antibodies and substrates having a value of Àlg K M falling within a particular range (K a = 10 3.5AE1.0 m À1 ).…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…Host-guest complexes have played a predominant role in the study of specific forces. Several experiments have been performed with different complementary molecules in the last years [368,[1118][1119][1120][1121][1122][1123][1124][1125][1126][1127][1128][1129]. It also has been shown, that force-displacement curves with functionalized tips can be employed to distinguish molecules of different chirality [1130,1131].…”