AFM protein-protein interactions within the EcoR124I molecular motor

Dynamic Force Spectroscopy (DFS), an Atomic Force Microscopy (AFM) technique, has been used to investigate the interaction between the HsdR subunit and the core methylase (MTase) of the Type I Restriction-Modification (R-M) enzyme EcoR124I. Such systems are of interest in bionanotechnology owing to their ability to translocate DNA, thus acting as molecular motors. Forces between a glutathione S-transferase (GST)-HsdR(PrrI) motor subunit attached to an AFM tip using a polyethylene gycol linker and the core MTase on poly-L-lysine pre-treated mica were measured at different loading rates. In the absence of an applied force, the position of energy barrier xdiss, bond dissociation rate kdiss(0) and lifetime of the bond (0) were calculated to be 1.35±0.17 nm, 0.16 s-1 and 6.3 s, respectively. The kdiss(0) value was a little lower than that obtained from magnetic tweezers (0.4 s-1), suggesting that the thermodynamic equilibrium may be affected by the presence of DNA. This work demonstrates that kinetic data concerning protein-protein interactions between subunits within Type I R-M enzymes are accessible via AFM. Such information is important for structure elucidation and the development of nanodevices.