Novel mechanistic insights into the pressure and temperature stabilization of enzymes

Abstract

Pressure perturbation calorimetry (PPC) was used to determine the volumetric properties for a diverse set of globular proteins, including six ancestral thioredoxins (Trx). The thermal expansion coefficient in the native state as a function of temperature (αN (T)) was shown to be very different for the studied proteins. After eliminating various structural and thermodynamic factors it was concluded that the differences in the native state expansivity function might be defined by the overall tertiary topology of the protein fold; a hypothesis that was supported by the data for the ancestral Trx. Also, the αN values were slightly different even for proteins with the same topology (Trx).

Lastly, the thermodynamic parameters of a group of acylphosphatase (Acp) proteins were determined using DSC and PPC. Based on the integration of the respective P-T profiles to obtain stability ranges, a negative trend of stability range with the Δα values was determined. There was also a weaker, but positive trend of stability range with the ΔV values. It was concluded that the Acps used a combination mechanism of stabilization by optimizing the Δα and ΔV parameters simultaneously. A larger set of Acp variants will be needed to further confirm these biophysical trends.

Pressure and temperature have important roles in the evolution of all organisms and their associated proteins. Both factors need to be considered when attempting to engineer more stable proteins for industrial and pharmaceutical applications. In this work, the thermodynamic properties of ancestral and modern proteins were extensively characterized to determine whether the ancestral proteins had unique, or in some way improved, parameters when compared to their modern homologues. The experimental findings, with a primary focus on the pressure-related (volumetric) parameters, could provide clues for engineering more stable proteins.

Additionally, the kinetics of folding and unfolding for the Trx were characterized to determine whether there was an optimization of kinetic properties over the evolutionary time. Based on Chevron plots, in which the log of the apparent kinetic rates is plotted against chemical denaturant concentration, we concluded that the folding kinetics were relatively conserved, but the unfolding kinetics were not. Unfolding kinetics did not correlate with evolutionary time, but correlated well with the denaturation temperature (Tm) and overall protein thermodynamic stability (ΔG). This implied that unfolding kinetics dictate the overall stability, at least for this particular Trx fold.

To investigate the thermodynamics further, a larger set of Trx was considered, comprised of ancestral and existing Trx. Differential scanning calorimetry (DSC) experiments were performed to obtain the temperature-related thermodynamic parameters and were supplemented by PPC experiments to evaluate the volumetric parameters. Pressure-temperature (P-T) profiles were constructed at equilibrium. The P-T ranges, or stability ranges, were determined by integrating the areas under these P-T profiles. The results indicate that ancestral Trx generally have improved stability ranges compared to those of modern Trx. This increase in stability range appears to originate from increased volume change upon unfolding (ΔV). Such a novel mechanism of stabilization has not been previously derived experimentally for any other proteins. There was no correlation of stability range with the change in the thermal expansion coefficient upon unfolding (Δα).