Dynamic Force Spectroscopy Analysis on the Redox States of Protein Disulphide Bonds.

Abstract

An emerging concept in chemical biology is that protein function that can be regulated by the redox state of disulphide bonds. This chapter describes the dynamic force spectroscopy method for analyzing redox regulation of receptor-ligand interactions at the surface of living cells. 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 (10-12N), ~3nm (10-9 m), and ~0.5ms (10-3s) in force, spatial, and temporal resolution. As a test bed model, we use the BFP to examine the autoregulation of von Willebrand factor function by a disulphide bond switch in its A2 domain. With the survival frequency analysis on measured bond lifetimes, we can identify distinct states of VWF binding kinetics and correlate with redox states of its A2 disulphide bond validated by mass spectrometry. The methodologies and analytical frameworks can be used to study other membrane receptor-ligand interactions under redox regulation.