TY - JOUR
T1 - Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates
AU - Schwesinger, Falk
AU - Ros, Robert
AU - Strunz, Torsten
AU - Anselmetti, Dario
AU - Güntherodt, Hans Joachim
AU - Honegger, Annemarie
AU - Jermutus, Lutz
AU - Tiefenauer, Louis
AU - Plückthun, Andreas
PY - 2000/8/29
Y1 - 2000/8/29
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0034730166&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034730166&partnerID=8YFLogxK
U2 - 10.1073/pnas.97.18.9972
DO - 10.1073/pnas.97.18.9972
M3 - Article
C2 - 10963664
AN - SCOPUS:0034730166
SN - 0027-8424
VL - 97
SP - 9972
EP - 9977
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 18
ER -