Protein Nanomechanics and Intermolecular Forces.

Traditionally, adhesion interactions have been studied in reversible equilibrium conditions. However, in vivo, these interactions 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. (1) The capabilities of these methods to increase our understanding on biological processes have been clearly demonstrated by a recent study on the dynamics of the interactions between individual living S. epidermidis cells and single Fn molecules performed using the SFM. (2) The energy landscape of the binding/unbinding process was mapped and the association and dissociation rate constants of the binding pair were measured at the single molecule level. The same approach has been also applied to study of the interactions of synthetic polymers or of DNA with inorganic surfaces. It proved to be capable of mapping the conformation assumed by the single polymeric molecules upon their adsorption on the substrate. (3,4) By means of the same single-molecule manipulation methods one can also stretch individual protein molecules. These experiments make it possible to investigate the forces that hold together the structure of proteins, and, at the same time, also to study how an external force unfolds and drives them 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. Angiostatin is one of them. It is composed of five kringle domains, each of them with three internal disulfide bonds. By the single-molecule force spectroscopy methodology we demonstrated how the redox state of these disulphide bonds control the topology and the mechanical properties of angiostatin. (5) The same methodology has made it possible to mimick the mechanical role of angiostatin in the reducing conditions met by this protein on the surface of a tumor cell and, under this basis, to propose a new mechanochemical approach to the antitumor activity of angiostatin. (6) (1) Samorì, B. (2000) Stretching single molecules along unbinding and unfolding pathways with the scanning force microscope. Chemistry, 6, 4249-4255. (2) Bustanji Y., Arciola C.R., Conti M, Mandello E., L. Montanaro, Samorì B. (2003) Dynamics of the Interaction between a Fibronectin Molecule and a Living Bacterium under Mechanical Force Proc. Natl. Acad. Sci. (USA) 100: 13292-13297 (3) M. Conti, Y. Bustanji, G. Falini, P. Ferruti, S. Stefoni, B. Samori’(2001) The desorption process of macromolecules adsorbed on interfaces:the force spectroscopy approach ChemPhysChem 2, 610-613 (4) B. Samorì (1998) Stretching, tearing and dissecting single molecules of DNA Angew. Chem. Int. Ed. Engl., 37, 2198-2200 (5) Bustanji, Y. and Samorì, B. (2002) The Mechanical Properties of Human Angiostatin can be Modulated by its Disulphide Bonds: a Single Molecule Force Spectroscopy Study Angew. Chem. Int. Ed., 41, 1546-1548. (6) Grandi F., Guarguaglini G., Sandal M., Samorì B. (2004) Angiostatin as a Mechanical Switch in Angiogenesis ( in publication)