The Molecular Underpinnings Of Thermostabilization Of Hyperthermophilic Protein Ssh10b

A statistical research showed that comparing the structural parameters between hyperthermophilic proteins and mesophilic ones, the only generally observed rule is an increase in the number of salt bridges and charged amino acid residues with increasing growth temperature. However, estimates of the energy contribution of electrostatic interaction to protein stability have led to conflicting conclusions. In this work, we took the hyperthermophilic protein Ssh10b as model, made some researches mainly focused on the relationships between enhanced protein stability and salt bridges/charged residues.Theoretical research showed that salt bridges are extremely resilient to temperature increases and thus are specially suited to promoting protein stability at high temperatures. However, so far there are no reports of experiments to measure the stability contribution of salt bridges at high temperatures and to provide evidence for these theoretical predictions. In this work, a double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in protein Ssh10b. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures.Moreover, comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the direct contribution to protein stability of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.In this work, we also proposed through theoretical analysis that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability. Acid/base titration demonstrated that relative to the unfolded state, the pKa values of many ionizable residues are shifted in the native Ssh10b. Theoretical prediction suggested that the pKa values of some alkali residues are downshifted in the native state, which should result in that at neutral pH region, the native Ssh10b is existed as a conformation ensemble with these ionizable residues in more disordered acid/base equilibrium. Theoretical calculation indicated that the increased conformational entropy in native state induced by pKa shifts at neutral pH region could stabilize the protein greatly.In addition, in order to study the stability contribution of the proline residues, in this work, we expressed and purified a series of Ssh10b mutants about proline, made correlative unfolding experiments in detail. We proposed a new unfolding mechanism including proline isomerization. According to this model, the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between the cis- and trans- isomers in both unfolded and folded states, agreeing well with unfolding experimental results. The results also demonstrated that the increased conformational entropy due to two native forms induced by proline isomerization contributes to stabilize the folded protein, providing experimental evidence indirectly for the viewpoint that the increase of conformational entropy in the native state due to pKa shifts of ionizable residues could enhance protein stability.