Abstract
Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Here we 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.