Directed Evolution of the Mechanical Properties of Protein Interactions

Abstract

Mechanical forces are ubiquitous in biological systems. Many physiological and pathological phenomena occur under the influence of hydrodynamic or cell adhesive forces, including processes such as antibody selection, cell detachment and bacterial adhesion to human extracellular matrix. It is now widely appreciated that protein interactions under the influence of force exhibit altered kinetic parameters and that mechanical properties of the interaction are independent of thermodynamic stability. These mechanical properties govern how protein complexes respond to applied mechanical stress, but their origins are poorly understood from a protein structure perspective. Current strategies for in vitro development of therapeutic and biotechnologically relevant binding proteins do not take the non-equilibrium response to force into account. The general lack of straightforward and high throughput methods to apply quantifiable mechanical forces to recombinant protein complexes precludes the use of directed evolution strategies. Moreover, it results in a shortage of data essential for the formulation of general principles that determine the mechanical strength of protein interactions, hindering rational design efforts to develop proteins with tuned mechanical properties. In this thesis, we address this problem by developing a high throughput selection platform that uses shear stress as an evolutionary filter to allow for the directed evolution of mechanostable protein interactions. This is achieved by exploiting the relationship between yeast cell adhesion strength and the molecular mechanostability of the complexes that mediate it. Using a spinning disk hydrodynamic shear-based assay, we successfully selected monomeric streptavidin mutants that mediated increased yeast adhesion under force, some of which also presented enhanced resistance to unbinding at the single molecule level. We further improved our selection platform by combining it with a yeast titratable display system, to improve the mechanostability of a cohesin-dockerin pair, through allosteric regulation and by altering the binding interface, in independent selection campaigns. Selection of both libraries yielded dockerin variants that showed increased complex dissociation through pathways capable of dissipating more energy, delaying complex rupture under force. The proposed shear-based yeast selection platform can become a powerful technology to identify proteins that mediate mechanically stable interactions and impact varied fields from biotechnology, biotherapeutics and biomaterials science. Furthermore, the acquisition of relevant data to help inform structural determinants of the mechanostability of protein interactions, represents a good learning opportunity at the level of fundamental research.