Role of Salt Bridges in Ubiquitin in Modulating Thermodynamic, Kinetic and Mechanical Stabilities

Abstract

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence. Ubiquitin a post translational modifier is mainly noted for its function in intracellular protein degradation has two salt bridges: one relatively exposed (SB1:K11-E34) and the other buried (SB2:K27-D52). Here we investigate the role of charged residues and hydrophobic interactions in protein folding by mutating salt-bridge residues in ubiquitin with hydrophobic residues. The thermodynamic stability of SB1 null variant is increased whereas the stability of the SB2 null variant is slightly reduced. We prepared a double salt bridge (DB) null variant and its thermodynamic stability is an additive effect of the individual salt bridges. Kinetic experiments reveal that that all the salt-bridge null variants fold through a more stable intermediate with relatively faster folding rates. The SB2 null variant has a highly stabilized unfolding transition state (TS) and a slightly destabilized native state, leading to its kinetic instability, whereas the kinetic stability of SB1 null variant is not compromised as its TS and native state are stabilized to a similar extent. The TS stabilization is also additive for the DB null variant having the most stabilized TS and high kinetic instability. Our results underscore the importance of kinetic stability in optimizing protein energy landscape. Our study establishes the fact that the TSs can be stabilized by hydrophobic residues in the place of buried charged residues and the effect can be even reverse of that observed for the native state. It further highlights the role of charged residues in the protein interior in modulating the thermodynamic and kinetic stabilities. In future we are going to investigate the role of salt bridges in tuning the mechanical properties of ubiquitin.