Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Deep subsurface environments can harbour high concentrations of dissolved ions, yet we know little about how this shapes the conditions for life. We know even less about how the combined effects of high pressure influence the way in which ions constrain the possibilities for life. One such ion is perchlorate, which is found in extreme environments on Earth and pervasively on Mars. We investigated the interactions of high pressure and high perchlorate concentrations on enzymatic activity. We demonstrate that high pressures increase α-chymotrypsin enzyme activity even in the presence of high perchlorate concentrations. Perchlorate salts were shown to shift the folded α-chymotrypsin phase space to lower temperatures and pressures. The results presented here may suggest that high pressures increase the habitability of environments under perchlorate stress. Therefore, deep subsurface environments that combine these stressors, potentially including the subsurface of Mars, may be more habitable than previously thought.

Lipid membranes are a key component of contemporary living systems and are thought to have been essential to the origin of life. Most research on membranes has focused on situations...

High pressure deep subsurface environments of Mars may harbor high concentrations of dissolved salts, such as perchlorates, yet we know little about how these salts influence the conditions for life, particularly in combination with high hydrostatic pressure. We investigated the effects of high magnesium perchlorate concentrations compared to sodium and magnesium chloride salts and high pressure on the conformational dynamics and stability of double-stranded B-DNA and, as a representative of a non-canonical DNA structure, a DNA-hairpin (HP), whose structure is known to be rather pressure-sensitive. To this end, fluorescence spectroscopies including single-molecule FRET methodology were applied. Our results show that the stability both of the B-DNA as well as the DNA-HP is largely preserved at high pressures and high salt concentrations, including the presence of chaotropic perchlorates. The perchlorate anion has a small destabilizing effect compared to chloride, however. These results show that high pressures at the kbar level and perchlorate anions can modify the stability of nucleic acids, but that they do not represent a barrier to the gross stability of such molecules in conditions associated with the deep subsurface of Mars.

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.