A comprehensive understanding of ligand−protein interactions requires information about all thermodynamic parameters that describe the complexation reaction, and they should be able to provide the necessary information to understand the molecular forces that drive complex formation. Usually, binding studies are performed at ambient pressure conditions. However, in addition to using temperature variation to reveal enthalpic and entropic contributions to ligand binding, complementary pressure-dependent studies providing volumetric properties of the reaction can be beneficial. Changes in partial molar volume can inform about changes in packing and hydration upon ligand binding. Here, after a general discussion of pressure effects on ligand binding reactions, we present a comprehensive study of the effect of pressure and a widely used organic cosolvent, dimethyl sulfoxide (DMSO), on the binding of a small aromatic ligand, proflavine, to the enzyme α-chymotrypsin. We found that DMSO, which acts as a competitive inhibitor for proflavine, has a strong impact on the interaction process, resulting in a decrease of the binding constant. While the reaction performed in neat buffer is basically pressure insensitive, the partial molar volume of the complex in the presence of DMSO is larger compared with the uncomplexed state, rendering the binding constant markedly smaller upon pressurization. We also show that the magnitude and sign of the binding volume provide valuable information about the interaction mechanism and hydration changes, which is of particular interest when cosolvents are present.

Because organisms living in the deep sea and in the subseafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding...

Protein–ligand interactions are fundamental to all biochemical processes. Generally, these processes are studied at ambient temperature and pressure conditions. We investigated the binding of the small ligand 8-anilinonaphthalene-1-sulfonic acid (ANS) to the multifunctional protein bovine serum albumin (BSA) at ambient and low temperatures and at high pressure conditions, in the presence of ions associated with the surface and subsurface of Mars, including the chaotropic perchlorate ion. We found that salts such as magnesium chloride and sulfate only slightly affect the protein–ligand complex formation. In contrast, magnesium perchlorate strongly affects the interaction between ANS and BSA at the single site level, leading to a change in stoichiometry and strength of ligand binding. Interestingly, both a decrease in temperature and an increase in pressure favor the ligand binding process, resulting in a negative change in protein–ligand binding volume. This suggests that biochemical reactions that are fundamental for the regulation of biological processes are theoretically possible outside standard temperature and pressure conditions, such as in the harsh conditions of the Martian subsurface.

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions’ role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl2, MgSO4, and Mg(ClO4)2) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid–ligand interactions of the type studied here.