Mechanism of pressure-induced thermo stabilization of proteins: Studies ofglutamate dehydrogenases from the hyperthermophile Thermococcus litoralis

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutan ts (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthe rmophile Thermococcus litoralis. All three GDHs were stabilized, although t o different degrees, by the application of 500 atm. Interestingly, the degr ee of pressure stabilization correlated with GDH stability as well as the m agnitude of electrostatic repulsion created by residues at positions 138 an d 167. Thermoinactivation experiments also were performed in the presence o f trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermo stabilization followed the same order as pressure sta bilization. Previous studies suggested a mechanism whereby the enzyme adopt s a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the thre e GDHs allowed us to further confirm. and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that press ure stabilizes against thermoinactivation by shifting the equilibrium betwe en conformational substates of the GDH hexamer, thus inhibiting irreversibl e aggregation.