Dynamic Force Spectroscopy for Analysis of Multiple Disulfide-Bonded Protein States.

An emerging concept in chemical biology is that protein function can be regulated by the redox state of disulfide bonds. This chapter describes the dynamic force spectroscopy method for analyzing the redox regulation of distinct disulfide-bonded protein conformations. The main method described in this chapter is the biomembrane force probe (BFP), in which an ultrasoft human red blood cell is used as an ultrasensitive mechanical force probe. The BFP uses a high-speed camera and real-time imaging tracking techniques to characterize a single molecular bond with ~1 pN (10-12 N), ~3 nm (10-9 m), and ~0.5 ms (10-3 s) in force, spatial, and temporal resolution. As an exemplar model, we use the BFP to analyze the binding kinetics of distinct antithrombin conformations with varying degrees of reduced disulfide bonds under a range of applied forces. Through examination of measured bond lifetimes, we can identify a change in antithrombin binding affinity to standard heparin and correlate the binding kinetics with the overall proportion of reduced disulfide bonds in the antithrombin protein as validated by mass spectrometry. The methodologies described here offer a robust framework for examining other disulfide-bonded protein states.