SpyTag/SpyCatcher tether as a fingerprint and force marker in single-molecule force spectroscopy experiments

Single molecule force spectroscopy has emerged as a powerful tool to study protein folding dynamics, ligand-receptor interactions, and various mechanobiological processes. High force precision does not necessarily lead to high force accuracy, as the uncertainties in calibration can bring serious systematic errors. In the case of magnetic tweezers, accurate determination of the applied forces for short biomolecular tethers, by measuring thermal fluctuations of inhomogeneous magnetic beads, remains difficult. Here we address this challenge by showing that the SpyTag/SpyCatcher complex is not only a convenient and genetically encodable covalent linker but also an ideal molecular fingerprint and force marker in single molecule force spectroscopy experiments. By stretching the N-termini of both SpyCatcher and SpyTag, the complex unfolds locally up to the isopeptide bond position in an unzipping geometry, resulting in equilibrium transitions at similar to 30 pN with step sizes of similar to 3.4 nm. This mechanical feature can be used as the fingerprint to identify single-molecular events. Moreover, the transitions occur with a fast exchange rate and in a narrow force range. Therefore, the real applied forces can be determined accurately based on the force-dependent transitions. The equilibrium forces are insensitive to buffer conditions and temperature, making the calibration applicable to many complicated experimental systems. We provide an example to calibrate protein unfolding forces using this force marker and expect that this method can greatly simplify force calibration in single-molecule force spectroscopy experiments and improve the force accuracy.

Automated summary

Single molecule force spectroscopy is a powerful tool for studying protein folding dynamics and other mechanobiological processes, but high force precision does not guarantee high force accuracy. The challenge in magnetic tweezers lies in accurately determining applied forces due to calibration uncertainties.