Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.
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.
Schwesinger
F
F
Biochemisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.
Ros
R
R
Strunz
T
T
Anselmetti
D
D
Güntherodt
H J
HJ
Honegger
A
A
Jermutus
L
L
Tiefenauer
L
L
Pluckthun
A
A
D019943:Amino Acid Substitution; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000648:ultrastructure; D001666:Binding Sites, Antibody; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000648:ultrastructure; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C030864:5-aminofluorescein; D016650:Fluorescein-5-isothiocyanate