Experiment-guided thermodynamic simulations on reversible two-state proteins: implications for protein thermostability Original Research Article

Here, we perform protein thermodynamic simulations within a set of boundary conditions, effectively blanketing the experimental data. The thermodynamic parameters, melting temperature (TG), enthalpy change at the melting temperature (ΔHG) and heat capacity change (ΔCp) were systematically varied over the experimentally observed ranges for small single domain reversible two-state proteins. Parameter sets that satisfy the Gibbs–Helmholtz equation and yield a temperature of maximal stability (TS) around room temperature were selected. The results were divided into three categories by arbitrarily chosen TG ranges. The TG ranges in these categories correspond to typical values of the melting temperatures observed for the majority of the proteins from mesophilic, thermophilic and hyperthermophilic organisms. As expected, ΔCp values tend to be high in mesophiles and low in hyperthermophiles. An increase in TG is accompanied by an up-shift and broadening of the protein stability curves, however, with a large scatter. Furthermore, the simulations reveal that the average ΔHG increases with TG up to ∼360 K and becomes constant thereafter. ΔCp decreases with TG with different rates before and after ∼360 K. This provides further justification for the separate grouping of proteins into thermophiles and hyperthermophiles to assess their thermodynamic differences. This analysis of the Gibbs–Helmholtz equation has allowed us to study the interdependence of the thermodynamic parameters TG, ΔHG and ΔCp and their derivatives in a more rigorous way than possible by the limited experimental protein thermodynamics data available in the literature. The results provide new insights into protein thermostability and suggest potential strategies for its manipulation.