Single-Molecule Force Spectroscopy of Isolated and Aggregated Fibronectin Proteins on Negatively Charged Surfaces in Aqueous Liquids

Plasma fibronectin (FN) was adsorbed to negatively charged surfaces, and the mechanical behavior of both isolated and aggregated FN molecules was observed using molecular force spectroscopy. Images of FN molecules show that the isolated proteins are already partially denatured, and mechanically pulling on them yields force transitions at distance intervals significantly shorter than the domains' contour lengths. Only when FN was aggregated on the surface did force transitions occur at length intervals corresponding to Type III domain lengths. Apparently, FN's density on the surface plays a critical role in protein stabilization. The dependence of the transition forces on the loading rates was also measured and modeled. In measurements done on single proteins in aggregates, one barrier in the direction of the applied force was observed, which arose from domain denaturation; however, studies of isolated, single molecules revealed two barriers, where both arise from protein−surface interactions.