Probing Intermolecular Interactions At The Single Molecule Level: The Dynamic Force Spectroscopy Approach.

Traditionally, protein-protein adhesion interactions have been studied in reversible equilibrium conditions. However, in vivo, these interactions might take place under significant shear forces with pulling rates that are much faster than the relaxation rates of the binding pair, and thus the binding/unbinding process occurs under non-equilibrium, irreversible conditions. The development of single-molecule manipulation methods based on the Scanning Force Microscopy (SFM) and on the Optical Tweezers, has made it possible to investigate the dynamics of these processes under non-equilibrium conditions and to measure their force-dependent dissociation kinetics. By means of the same single-molecule manipulation methods one can investigate the forces that hold together the structure of proteins and the interactions that drive their folding. In the length-clamp mode, these methods make it possible to study how an external force unfolds and drives a protein towards non-equilibrium conformations. These studies are particularly meaningful when the proteins investigated are involved in-vivo in transport and mechanical processes. In this case the force spectroscopy experiments do simulate, at the single molecule level, the same mechanical role exerted by those proteins in-vivo. In the force-clamp modes, these manipulation methods make it possible to record the entire folding trajectory of a single protein as a function of time and to resolve its folding pathway.