High-Speed Force Spectroscopy for Single Protein Unfolding

: Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain.