High-Speed Force Spectroscopy for Single Protein Unfolding

Methods Mol Biol. 2018;1814:243-264. doi: 10.1007/978-1-4939-8591-3_15.


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.

Keywords: Atomic force microscopy; Cohesion; Dockerin; High-speed force spectroscopy; Protein unfolding; Titin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calibration
  • Connectin / chemistry*
  • Data Analysis
  • Microscopy, Atomic Force / methods*
  • Protein Unfolding*


  • Connectin