Liquid–liquid phase separation (LLPS) of proteins and other biomolecules play a critical role in the organization of extracellular materials and membrane‐less compartmentalization of intra‐organismal spaces through the formation of condensates. Structural properties of such mesoscopic droplet‐like states were studied by spectroscopy, microscopy, and other biophysical techniques. The temperature dependence of biomolecular LLPS has been studied extensively, indicating that phase‐separated condensed states of proteins can be stabilized or destabilized by increasing temperature. In contrast, the physical and biological significance of hydrostatic pressure on LLPS is less appreciated. Summarized here are recent investigations of protein LLPS under pressures up to the kbar‐regime. Strikingly, for the cases studied thus far, LLPSs of both globular proteins and intrinsically disordered proteins/regions are typically more sensitive to pressure than the folding of proteins, suggesting that organisms inhabiting the deep sea and sub‐seafloor sediments, under pressures up to 1 kbar and beyond, have to mitigate this pressure‐sensitivity to avoid unwanted destabilization of their functional biomolecular condensates. Interestingly, we found that trimethylamine‐N‐oxide (TMAO), an osmolyte upregulated in deep‐sea fish, can significantly stabilize protein droplets under pressure, pointing to another adaptive advantage for increased TMAO concentrations in deep‐sea organisms besides the osmolyte's stabilizing effect against protein unfolding. As life on Earth might have originated in the deep sea, pressure‐dependent LLPS is pertinent to questions regarding prebiotic proto‐cells. Herein, we offer a conceptual framework for rationalizing the recent experimental findings and present an outline of the basic thermodynamics of temperature‐, pressure‐, and osmolyte‐dependent LLPS as well as a molecular‐level statistical mechanics picture in terms of solvent‐mediated interactions and void volumes.

Biomolecular condensates can be functional (e.g., as membrane-less organelles) or dysfunctional (e.g., as precursors to pathological protein aggregates). A major physical underpinning of biomolecular condensates is liquid–liquid phase separation (LLPS) of proteins and nucleic acids. Here we investigate the effects of temperature and pressure on the LLPS of the eye-lens protein γ-crystallin using UV/vis and IR absorption, fluorescence spectroscopy, and light microscopy to characterize the mesoscopic phase states. Quite unexpectedly, the LLPS of γ-crystallin is much more sensitive to pressure than folded states of globular proteins. At low temperatures, the phase-separated droplets of γ-crystallin dissolve into a homogeneous solution at as low as ∼0.1 kbar whereas proteins typically unfold above ∼3 kbar. This observation suggests, in general, that organisms thriving under high-pressure conditions in the deep sea, with pressure of up to 1 kbar, have to cope with this pressure sensitivity of biomolecular condensates to avoid detrimental impacts to their physiology. Interestingly, our experiments demonstrate that trimethylamine-N-oxide, an osmolyte upregulated in deep-sea fish, significantly enhances the stability of the condensed protein droplets, pointing to a previously unrecognized aspect of the adaptive advantage of increased concentrations of osmolytes in deep-sea organisms. As the birth place of life on earth could have been the deep sea, studies of pressure effects on LLPS as presented here are relevant to the possible formation of protocells under prebiotic conditions. A physical framework to conceptualize our observations and further ramifications of biomolecular LLPS at low temperatures and high hydrostatic pressures is discussed.

We investigated the combined effects of temperature and pressure on liquid-liquid phase separation (LLPS) phenomena of α-elastin up to the multi-kbar regime. FT-IR spectroscopy, CD, UV/Vis absorption, phase-contrast light and fluorescence microscopy techniques were employed to reveal structural changes and mesoscopic phase states of the system. A novel pressure-induced reentrant LLPS was observed in the intermediate temperature range. A molecular-level picture, in particular on the role of hydrophobic interactions, hydration, and void volume in controlling LLPS phenomena is presented. The potential role of the LLPS phenomena in the development of early cellular compartmentalization is discussed, which might have started in the deep sea, where pressures up to the kbar level are encountered.

Biomolecular condensates formed by liquid–liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo. One of the most common experiments in biochemistry, biophysics, medicinal chemistry, and cellular biology is testing whether a ligand binds to a protein 1-5. Protein-ligand recognition and interaction are fundamental to many events essential to life, such as self-replication, metabolism and signal transduction. Furthermore, elucidating the nature of the forces involved in the binding processes is prerequisite for the development of new and more effective drugs in medical applications. In spite of its apparent importance, many aspects of ligand binding have not been fully explored, yet. Commonly, binding studies are carried out in dilute buffer solution and at ambient temperature and pressure. But the interior of biological cells is enriched with numerous macromolecules, such as proteins and nucleic acids, forming a highly crowded environment. Crowding affects molecular diffusion, conformation, dynamics and kinetics as well as the hydration properties of proteins 6-9. Further, biological cells need to orchestrate their biochemical reactions in space and time. The modulation and regulation of such processes is achieved through the compartmentalization of the cellular milieu. Besides lipid bilayer membranes, non-membrane bound compartments lacking a surrounding lipid bilayer and consisting of phase-separated liquid-like droplets have been shown to be of similar importance in recent years 10,11. Such membrane-less droplet-like compartments, also denoted biomolecular condensates, ar...