Deciphering the Properties of EspA Protein by Single Molecule Force Spectroscopy in Search for Novel Nano-Fibers

Hila Nadler, Chemical Engineering, Ben Gurion University, Rishon LeZion, Israel

The search of novel materials with enforced properties met a new direction in the studies of biological fibers. Spider silk, for instance, have been studied intensively due to their nearly mythical mechanical properties. However, technical challenges are still limiting the mass production of these biological fibers. Hence, protein based fibers from bacterial origin can resolve these difficulties. EspA (E.Coli secreted protein A) is a protein that self-assemble to form a bacterial external syringe-like filament, which is a major part of the type III secretion system (T3SS) utilized by bacteria to inject virulence factors into cells that the bacteria infects. This long EspA filament penetrates the thick mucus barrier that protects the epithelial cells in the intestinal tract to facilitate the initial attachment of the bacterial pathogens to the host cell. Considering the mechanical shear stresses induced by the mucosal flow, the EspA filament possess remarkable elastic endurance. To date, very little is known about the mechanical properties of the EspA filament. Moreover, even less is known on the EspA protein structure and properties since it is highly reactive. Here we use Single Molecule Force Spectroscopy (SMFS) to study the structural and mechanical properties of a single EspA protein. By applying controlled load unto the molecule, we record its dynamical response while unfolding. From these trajectories, we provide details that relate to the protein mechanical stability through its free energy landscape. The novel information obtained in this methodology will provide insights on EspA assembly and mechanical properties as well as ways to control and manipulate the protein filamentous structure. Moreover, the fact that EspA is a naturally secreted bacterial protein offers an easy platform for protein manipulation and large-scale production.