Enhancing the Mechanical Stability of Proteins through a Cocktail Approach

Abstract

Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Several strategies have been developed successfully to rationally regulate the mechanical stability of proteins. These strategies include rational control of the unfolding pathway by disulfide bond formation, improving hydrophobic packing, reconstruction of the force-bearing region of proteins, ligand binding, and engineered metal chelation. However, compared with the well-developed methods used to enhance the thermodynamic stability of proteins/enzymes, these methods to enhance the mechanical stability remain limited. Furthermore, these methods are only used one at a time, and the resulted enhancement in protein mechanical stability is also rather limited. Possible synergetic effects from more than one method remain largely unexplored. Here we use single molecule force spectroscopy techniques to demonstrate that it is feasible to use a ‘‘cocktail’’ approach for combining more than one approach to enhance significantly the mechanical stability of proteins in an additive fashion. As a proof of principle, we show that metal chelation and protein-protein interaction can be combined to enhance the unfolding force of a protein to ∼450 pN, which is >3 times of its original value. This is also higher than the mechanical stability of most of proteins studied so far. We also extend such a cocktail concept to combine two different metal chelation sites to enhance protein mechanical stability. This approach opens new avenues to efficiently regulating the mechanical properties of proteins, and should be applicable to a wide range of elastomeric proteins.