title: Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis.
abstract: 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 mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.
authors.affiliation: Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Sun
authors.forename: R
authors.initials: R
authors.lastname: Caillot
authors.forename: G
authors.initials: G
authors.lastname: Mak
authors.forename: F T
authors.initials: FT
authors.lastname: Robb
authors.forename: D S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D011312:Pressure; D020836:Protein Structure, Quaternary; D019712:Thermococcus / Q000201:enzymology*; D014199:Trehalose / Q000378:metabolism
chemical_list: D014199:Trehalose; D005969:Glutamate Dehydrogenase