Native Salt Bridges Are a Key Regulator of Ubiquitin's Mechanical Stability.

Although it is known that various intramolecular interactions determine protein mechanical stability, a detailed molecular-level understanding of the key regulators of protein mechanical stability is still lacking. Here, we present evidence for salt bridges in ubiquitin as important intramolecular interactions that can affect protein mechanical stability. Ubiquitin has two salt bridges: one relatively surface-exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Ubiquitin is a reversible post-translational modifier and is stable mechanically ( F avg u = 185 pN). On breaking SB1, the mechanical stability of ubiquitin is slightly enhanced ( F avg u = 193 pN). In contrast, the mechanical stability significantly decreased upon breaking SB2 ( F avg u = 158 pN). These results suggest that SB1 are SB2 are regulators of the mechanical stability of ubiquitin. Interestingly, the mechanical stability decreased further ( F avg u = 145 pN) for the double salt bridge (DB) null variant. Monte Carlo simulations elucidate that the main regulating factor is the spontaneous unfolding rate constant ( k u 0 ), being the highest for the DB null variant followed by the SB2 null variant, and it remains unaltered for the SB1 null variant, while the native-to-transition-state distance ( x u ) remains unchanged. Our study provides mechanistic understanding on how two native salt bridges can independently regulate the mechanical stability in a protein, which has implications in designing protein-based robust biomaterials in the future.