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.
Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.
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