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Updated: Mar 8, 2026

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Published on: February 28, 2019
Single-molecule force spectroscopy of fast reversible bonds
Johanna Blass1, Marcel Albrecht2, Gerhard Wenz2
1INM - Leibniz Institute for New Materials and Physics Department, Saarland University, Campus D2 2, 66123 Saabrücken, Germany. roland.bennewitz@leibniz-inm.de.
Unbinding force in single-molecule studies depends on force probe stiffness, not linker stiffness, for fast reversible bonds. This finding aids in understanding molecular interactions and binding energies.
Area of Science:
- Biophysics
- Chemical Physics
- Materials Science
Background:
- Single-molecule force spectroscopy quantifies molecular bond strength via unbinding force.
- Fast reversible bonds present unique challenges in accurately measuring interaction strength.
Purpose of the Study:
- To analyze the dynamics of force probe and molecular linker in thermodynamic equilibrium for fast reversible bonds.
- To systematically investigate the influence of cantilever and linker dynamics on unbinding force measurements.
Main Methods:
- Atomic force spectroscopy was employed to measure the unbinding force of single cyclodextrin inclusion complexes.
- Experiments utilized various molecular linkers and systematically varied force probe stiffness.
Main Results:
- Unbinding force is not unique but scales with the square root of force probe stiffness.
- Molecular linker stiffness had minimal impact on the measured unbinding force.
- Fast rebinding kinetics (order of 10^6 kHz) were observed, influenced by cantilever dynamics acting as a low-pass filter.
Conclusions:
- The observed scaling is explained by an effective potential arising from fast linker fluctuations and rebinding kinetics.
- Binding energy can be estimated from unbinding force versus cantilever stiffness, though 3D modeling uncertainties exist.
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