The effect of pressure on chemical equilibria is determined by the reaction volume \(\Delta V\) defined as the difference in partial molar volume between the product \(V_P\) and reactant \(V_R\) states and determined experimentally as the pressure derivative of the Gibbs energy of reaction. \(\Delta V = V_P – V_R = \frac{\partial \Delta G}{\partial p}\) Equilibria with negative \(\Delta V\) will favour the product state upon application of pressure, whereas equilibria with positive \(\Delta V\) will favour the reactant state. Protein denaturation is a process with a negative \(\Delta V\), which means that increased pressure typically results in dissociation of protein subunits, local conformational changes or even large-scale unfolding. Despite this, there are many marine organisms that thrive in the high pressure environment of the deep sea, thanks to evolutionary adaptations that stabilize their proteins against the denaturing effect of pressure. One example is the enzyme lactate dehydrogenase (LDH), which has been observed to have 21 amino acid substitutions in the deep sea fish Coryphaenoides armatus, compared with the shallow-water fish Gadus morhua.[1] However, the mechanics of how these substitutions contribute to high-pressure stability in C. armatus are unknown. One of the possible mechanisms an increase in the \(\Delta V\) of denaturation, disfavouring the denatured state at high pressure. Insight into the volumetric properties of these amino acid substitutions may be beneficial for the development of enzymes that can catalyze reactions under high-pressure. We used the molecular dynamics-based Archimedean Displacement technique[2] to investigate the effect of amino acid substitution on the denaturation \(\Delta V\) of LDH. The volume of the protein is taken to be the difference in volume between a simulated system of solvent containing the protein and a simulated system containing the same quantity of solvent without the protein. Since LDH is a tetramer, denaturation can occur through two mechanisms: dissociation of the monomer subunits or complete unfolding of the protein. Here, we present results for the \(\Delta \Delta V\) of each denaturation mechanism in pure water, as well as in the presence of the stabilizing agent trimethylamine-N-oxide.

[1] A. A. Brindley et al, PLoS ONE, 3, e2042(2008) [2] H. Wiebe et al. J. Phys. Chem. C 116, 2240–2245 (2012)