Engineering mechanical strength and resistance to fatigue of a nanostructured protein material through genetic removal of electrostatic repulsions

Protein nanoarrays and other protein-based nanostructures are being developed for many biomedical and technological applications. However, many protein assemblies are prone to disruption under load, and susceptible to material fatigue. Acquisition of a fundamental knowledge on the relationship between structure and mechanical properties of protein assemblies may guide the engineering of protein nanostructures with a higher strength and resistance to fatigue. The capsid protein (CA) of the human immunodeficiency virus can self-assemble into a single molecule-thick, flexible nanoarray that can coat large surfaces. In this study, a genetic engineering strategy was used to individually remove in the CA protein array the side chain of 7 amino acid residues per subunit involved in intermolecular interactions. The effects of eliminating those side chains, and their intermolecular interactions, on the equilibrium dynamics, stiffness, strength, and resistance to fatigue of the CA nanoarray were quantified using atomic force microscopy. The results revealed that removal of different types of attractive intermolecular interactions (van der Waals contacts, hydrogen bonds, and/or ionic bonds), increased the conformational flexibility of the protein array, and decreased its stiffness and strength. In contrast, removal of a naturally occurring, repulsive charge-charge intermolecular interaction in the nanoarray actually led to dramatic increases in strength against point loads (by ∼60%) and resistance to fatigue (by ∼50%), without increasing its stiffness. These findings suggest a general genetic strategy to increase the intrinsic strength and resistance to fatigue of nanostructured protein materials, based on the optimization of the ionic interactions between subunits.