Pressure-Sensitive and Osmolyte-Modulated Liquid-Liquid Phase

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Pressure-Sensitive and Osmolyte-Modulated LiquidLiquid Phase Separation of Eye-Lens Gamma-Crystallins Süleyman Cinar, Hasan Cinar, Hue Sun Chan, and Roland Winter J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Journal of the American Chemical Society

Pressure-Sensitive and Osmolyte-Modulated Liquid-Liquid Phase Separation of Eye-Lens Gamma-Crystallins Süleyman Cinar [a], Hasan Cinar [a], Hue Sun Chan [b], and Roland Winter [a]*

[a] Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology TU Dortmund, Otto-Hahn-Strasse 4a, 44227 Dortmund (Germany) *E-mail: [email protected] [b] Departments of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8 (Canada) Keywords: high pressure • liquid-liquid phase separation • crystallins • TMAO Abstract: Biomolecular condensates can be functional (e.g., as "membrane-less organelles") or dysfunctional (e.g., as precursor 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 at highpressure conditions in the deep sea, with pressure up to 1 kbar, have to cope with this pressure-sensitivity of biomolecular condensates to avoid detrimental impact on 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 under low temperatures and high hydrostatic pressures is discussed. 1. Introduction Within and around biological cells, biomolecules carry out complex physico-chemical processes that involve a large number of interacting entities. The environments in which these processes occur---especially that inside the cell---are not well-mixed homogeneous solutions.1-3 Certain biomolecules in the cell---especially those in the eukaryotic cell---operate in well-organized restricted spatial regions known as organelles. Now, in addition to wellknown organelles bound by lipid membranes such as the mitochondria and the nucleus, mounting evidence has emerged in recent years that liquid-liquid phase separation (LLPS) of proteins and nucleic acids can play critical roles in the assembly of functional compartments not encapsulated by membranes. Examples of such “membrane-less organelles” include cytoplasmic granules, nucleoli, clusters of proteins involved in signaling,4-9 and postsynaptic densities.10 Many of these intracellular bodies resemble liquid-like droplets that are selectively enriched in certain biomacromolecules including specific species of proteins and nucleic acids. As such, they are instances of "biomolecular condensates", which is a general designation that encompasses similar condensed-phase structures in the extracellular milieu.11 One advantage of membrane-less compartments is that their biological function can

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be switched on and off relatively rapidly by regulating the LLPS that underlies the formation and dissolution of the condensed phase. Notably, while LLPS of proteins are important for normal biological function, unregulated or mis-regulated LLPS can lead to diseases, as exemplified by cold cataract produced by the undesired aggregation of γ-crystallin within the vitreous fluid of the eye, sickle-cell disease, and eventually to amyloidogenic diseases.10,12-14 Physics dictates that LLPS commences at macromolecular concentrations at which the favorable effective inter-macromolecule interactions overcome the entropic drive to keep the solution homogeneously mixed. For proteins, these interactions are governed by their amino acid sequences (which determines, among other properties, whether a protein can be folded and, if so, its folded structure and the surface interactions it entails) as well as the modulating solution conditions that impact the interactions involved. These interactions may include charge-charge,15,16 polar, π-related,17 hydrogen-bonding and hydrophobic interactions,18 resulting in rich and highly complex phase behaviors of proteins12 in aqueous systems where the structure of water and the modulating effects of cosolvents are known to depend intricately on temperature, pressure and solution composition.19,20 Inasmuch as temperature dependence is concerned, some proteins exhibit an upper critical solution temperature (UCST) in that LLPS occurs only below a critical temperature Tc (i.e., Tc = UCST, the macromolecular concentration at Tc is its critical concentration cc; see Fig. 1). Other proteins such as elastin which are more hydrophobic exhibit a lower critical solution temperature (LCST), in which case LLPS occurs at temperatures higher than Tc = LCST and the system remains homogeneous at lower temperatures (Fig. 1).21,22

(a)

(b)

Figure 1. Liquid-liquid phase separation (LLPS) of proteins. (a) Schematic temperatureconcentration phase diagram with a UCST and an LCST [●: critical points, (cc,Tc)]. Corresponding effects of pressure and cosolvents on LLPS are largely unknown. (b) Examples of experimentally observed LLPS with UCST: lysozyme (pH 4.5, 0.1 NaAc, 0.5 M NaCl),23 γIIIb-crystallin (pH 7.1, 0.1 M phosphate buffer).24 The temperature, pressure, and cosolvent dependences of LLPS likely contribute toward the in vivo and in-cell responses of biomolecular condensates to environmental stress. Pertinent model LLPS systems in vitro have been demonstrated to respond to changes in temperature, pH and salt concentration.8,9 By comparison, high hydrostatic pressure (HHP) as a stress factor for biomolecular LLPS is largely unexplored.25-31 Yet a large fraction of the Earth’s biosphere, in the deep sea as well as the sub-seafloor crust, is under HHP up to 1 kbar (100 MPa, ~1,000 atm) and beyond.32 HHP studies on biomolecular systems are thus necessary for understanding the physical basis of extant life in the deep sea, which might be the birth place of life on Earth.32 Aside from this direct relevance to biology, pressure serves as a useful probe for investigating biomolecular interactions. As


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increasing pressure favors states with lower volumes, high pressure can reveal low-volume configurational states that are functionally important but only sparsely populated under ambient conditions.25,33,34 Since partial molar volumes of biomolecules are sensitive to hydration, HHP studies provide important insights into biomolecular packing.25,35-38 Seeking progress in this context, we investigated effects of pressure on the LLPS of γ-crystallins, which belong to the family of highly homologous mammalian lens proteins and are particularly concentrated in the cytoplasm of the eye lens cells.38 From the cortex to the nucleus, their concentration increases, even up to several hundred mg mL-1, thereby defining the refractive index necessary for focusing light.39 In vertebrates, α-, β- and γ-crystallins are the main types of crystallin. The denser core of the lens is populated predominantly or even exclusively by γ-crystallins.40,41 Transparency is needed for the proper function of eye lens. However, severe concentration fluctuations or even LLPS and/or protein crystallization of crystallins can induce light scattering and hence lens opacity.12,40,42,43 Hence knowledge of the pressure-dependent LLPS properties of crystallins is important for understanding how their function is adapted to different pressure environments. Interestingly, highly concentrated solutions of crystallins are known to undergo LLPS at low temperatures, a process also referred to as coacervation (Fig. 1b); and their UCST has been shown to depend on the protein’s specific amino acid sequence.22,39,44,45 The study reported here was on human and rat γ-D-crystallin. Both are the product of the γ-D gene.46 The amino acid sequences of rat and human proteins are 85.1% identical. Their percentages of basic and acidic residues are the same, but the proportion of hydrophobic amino acids in rat γ-Dcrystallin is slightly higher. We have utilized UV/Vis, FTIR and fluorescence spectroscopy, turbidity measurements, and light microscopy in various high-pressure sample cells to study the structure and phase properties of these proteins, covering low temperatures and a range of pressures that are relevant to conditions encountered in the deep sea (experimental details in the SI). Cosolvents in the biological cell can affect the relative stabilities of biomolecular states. To better understand how they modulate effects of pressure on biomolecular LLPS, we extended our study of pressure-sensitive γ-D-crystallin LLPS to address the impact of trimethylamine-N-oxide (TMAO). TMAO is an osmolyte known to stabilize the folded native state of globular proteins because of favorable TMAO-water interactions and unfavorable TMAO-protein interactions, which lead to enhanced thermodynamic stabilities of protein configurational states with a lower solvent-accessible surface area (SASA).3,47,48,49 Interestingly, TMAO is upregulated in organisms thriving in the deep sea. For that reason, TMAO is believed to serve as a pressure counteractant, or a "piezolyte".50 The present investigation is the first to probe how TMAO might affect functional protein LLPS under HHP. 3. Results To ascertain whether γ-D-crystallin undergoes LLPS through association of individual natively folded molecules rather than through association of certain pressure-unfolded form of the protein, we checked if the native protein fold is retained up to the highest pressure used in our LLPS study by intrinsic tryptophan fluorescence spectra of 0.1 mg mL-1 γ-Dcrystallin (50 mM TRIS, 150 mM NaCl, pH 7.4, T = 4 °C). The data (Figure SI 1) show no changes in intensity or shift of the fluorescence band from 1 bar to 2.4 kbar, indicating that the protein remains in the native folded state for the entire pressure range. Complementary studies using Fourier-transform infrared (FTIR) spectroscopy, probing changes in secondary structure elements of the protein, were carried out as well. In agreement with the fluorescence studies, no structural changes were observed up to 3 kbar for the protein in the dilute phase. Further, pressures up to 3 kbar had also no effect on the secondary structure of the protein in the LLPS region at low temperature (Figure SI 2). Coacervation was examined by monitoring the turbidity (apparent absorption) through light scattering at 400 nm using a UV/Vis spectrometer (Figure SI 3). The temperature of the sample cell was controlled by an external water thermostat. The pressure-dependent measurements were carried out using a home-built high-pressure optical cell.22 Sapphire with a diameter of 20 mm and a thickness of 10 mm was used for the window. Pressure was



applied using a high-pressure hand pump and was measured by a pressure sensor. Fig. 2a shows the UV/Vis turbidity of human and rat γ-D-crystallin as a function of temperature under atmospheric pressure. The human γ-D-Crystallin at ambient pressure exhibits a temperatureinduced cloud point at ~3 °C. Above ~5 °C, a homogeneous phase ensued. The cloud point of rat γ-D-crystallin is slightly higher at ~6 oC and the corresponding transition to homogeneous solution is complete at 8 oC for the protein concentration used in the experiment. Pressure dependent UV/Vis data for human γ-D-crystallin in Fig. 2b indicate that increasing pressure from 1 bar to ∼125 bar for the neat human γ-D-crystallin solution at 4 °C leads to a homogeneous phase (transition midpoint at p1/2 ≈ 80 bar). For rat γ-D-crystallin, the transition at 4 °C and 7 °C is broader (the trend is similar to the temperature-induced transition in Fig. 2a), centered at ∼250 bar and completed at 450 bar. These observations are in line with literature data45 indicating that relatively small changes in amino acid composition/sequence can have a significant effect on the cloud-point temperature and reveal further that such changes can have a significant effect on the cloud-point pressure as well.

Figure 2. Temperature-, pressure-, and TMAO-dependent LLPS. UV/Vis absorption intensity (turbidity) at 400 nm of a 55 mg mL-1 solution of human and rat γ-D-crystallin (50 mM TRIS, 150 mM NaCl, pH 7.4) is measured as a function of (a) temperature and (b) pressure at selected temperatures. (c,d) Effect of 0.38 and 0.6 M TMAO on the temperature- and pressure-dependent turbidity of human γ-D-crystallin. The absorption data are normalized to their maximum values (absorbance = 1.0). Quite remarkably, the addition of TMAO leads to a drastic increase of the cloud-point temperature (cloud point is hereby defined by lack of turbidity): For a 55 mg mL-1 solution of human γ-D-crystallin, adding 0.38 M TMAO shifts the cloud point from ∼5 to ~15 °C, adding


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0.6 M TMAO shifts it to ~25 °C (Fig. 2c). TMAO also reduces the pressure sensitivity of the LLPS. The pressure-induced cloud point shifts from ∼125 to ~400 bar when 0.38 M TMAO is added. It shifts further to ~600 bar when 0.6 M TMAO is present (Fig. 2d). In order to directly visualize the formation and disappearance of droplets in the LLPS process, studies were carried out using a light microscope and a home-built high-pressure optical cell with flat diamond windows that can withstand pressures up to about 2 kbar (Figure SI 4). At low temperatures, droplets are formed in the solution, which then sediment to the bottom to form macroscopic liquid–liquid phase separated regions after prolonged durations, leading to a decrease of turbidity in the bulk phase. The objective's focus in the microscopy images shown in Fig. 3 was on the inner surface of the transparent cell.

Figure 3. Pressure-dependent droplet formation and dissolution. Selected snapshots of light microscopy of 55 mg mL-1 human γ-D-crystallin (50 mM TRIS, 150 mM NaCl, pH 7.4, T = 4°C) in the pressure range from 1 to 400 bar. The objective's focus in these images was on the inner window surface of the optical cell. Owing to adsorption of the droplets at the optical window, dissolution of droplets seems to occur at slightly larger pressures compared to the bulk scenario (Figs. 2 and SI 5). Consistent with the turbidity measurements (Figure 2), increasing pressure leads to the formation of a homogeneous phase. Figure 3 shows that the process is completed at ∼400 bar. After pressure release, liquid-liquid phase separation occurs again, i.e., the process is apparently fully reversible. In the bulk, droplet formation can be more easily detected in the depressurization direction, which takes place between about 300 and 50 bar for the neat buffer system (see movie M 1 and Figure SI 5). Also, a certain degree of hysteresis and time-dependence of the nucleation event is expected to lead to minor



differences in the transition pressures detected. Figure 4a provides corresponding data for rat γ-D-crystallin during pressure release, showing a higher onset pressure for droplet formation at 7 oC. As a representative example, Figue 4b shows our pressure-dependent microscopy data for human γ-D-crystallin in the presence of 0.6 M TMAO at 4 °C. Now the homogeneous phase is formed at ~500 bar. At these high TMAO concentrations, much fewer small droplets are seen at the beginning of the experiment (at ambient pressure, 1 bar) because apparently TMAO fosters coalescence of droplets.

Figure 4. TMAO modulates pressure-dependent LLPS. Light microscopy snapshots of 55 mg mL-1 (a) rat γ-D-crystallin (T = 7°C) in the absence of TMAO, and (b) human γ-D-crystallin (T = 4°C) in the presence of 0.6 M TMAO (50 mM TRIS, 150 mM NaCl, pH 7.4 for both). 3. Discussion A plausible physical rationalization of the pressure dependence of γ-crystallin LLPS is outlined in Figure 5. As noted above, since no changes in secondary structure are observed upon droplet formation, the LLPS is very likely a result of association of individual natively folded protein molecules or minimally perturbed versions thereof. In view of the charged and hydrophobic residues on the folded γ-D-crystallin surface (Figure 5b,c), the phase-separated condensed phase of γ-D-crystallin at low temperature and ambient pressure is expected to be stabilized by transient electrostatic, hydrophobic, and van der Waals interactions among neighboring protein molecules, likely via dynamic contacts among the charged and nonpolar patches51 on the protein surfaces (Figure 5d, left). Intuitive geometric considerations as well as implicit- and explicit-water simulations of small model compounds, peptides, and model globular proteins18,52-54 suggest that a large number of voids, i.e., water-free cavities, are harbored by the transiently touching protein molecules in the relatively condensed liquid-like droplets (Figure 5d, left). The situation is similar to the presence of void volumes in folded proteins, although the voids in the latter are essentially static whereas those in the γcrystallin droplets are envisioned to be dynamic. As for the destabilization of folded proteins,25-29 or the melting of voluminous water cages in bulk water at high pressures,20 the condensed droplet phase becomes unstable under high pressure since a reduction of void volume is achievable by a homogeneous dilute phase, which is also favored by a higher mixing entropy (Figure 5d, right). Because the present phase transition pressures are more than an order of magnitude smaller compared to those leading to protein unfolding,25-29 the transient void volume in the dense droplet phase is probably large, which is physically plausible in view of the irregular shapes of folded proteins and therefore a likely


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preponderance of packing defects. At the same time, for the same loss of cohesive interactions, the configurational entropy increase upon droplet dissolution is likely larger than the gain in conformational entropy when a globular protein unfolds because a droplet dissociates into multiple molecules, each of which possesses essentially independent translational freedom, whereas a protein chain remains connected even when it unfolds. This difference in configurational/conformational entropic free energy of the dissociated states may account as well for the reduced stability of protein droplets under pressure. Moreover, although hydrogen bonds are generally strengthened by pressure,25 studies of simple model systems18,55 suggest that high pressure destabilizes hydrophobic contacts relative to compact yet solvent-separated configurations. The latter trend also favors droplet dissolution at moderately—though not extremely—high pressures.22 Interestingly, TMAO can modulate the pressure dependence of γ-crystallin LLPS (Figure 5e). The detailed mechanism for the effect of TMAO on the distribution of biomolecular configurations remains to be elucidated because results from atomic simulation studies to date are highly model dependent.47,56-58 Nonetheless, leaving aside the possible intricate effects of patchy interactions (see above) on diffusion dynamics and the impact of TMAO on the spatial ranges of effective biomolecular interactions,51,59 it is generally agreed that TMAO stabilizes compact protein configurations because it interacts unfavorably with proteins, as evident from the fact that TMAO is depleted around protein surfaces.47,60 This tendency applies to both unfolded and folded surfaces. Whereas the degree of unfavorability depends on the amino acid residues involved, experimental evidence suggests that TMAO is excluded from both nonpolar/hydrophobic as well as polar surfaces of proteins.61 The presence of TMAO can therefore counteract droplet dissolution by pressure (Figure 5e) as observed in our experiments because the droplet (condensed) phase has less solvent-exposed protein surface and therefore fewer unfavorable interactions with TMAO than that entailed by the larger total solvent-exposed protein surface in the dispersed (dilute) phase. The same principle holds even if the condensed phase contains partially unfolded crystallin (Figures 5f,g). It is conceivable, a priori, that the crowded environment in the condensed phase may enable partial unfolding of individual proteins even when the solution conditions are strongly favorable for individual folded structures in the dilute phase. In such a hypothetical situation, the transient interactions among the folded and disordered parts of multiple protein molecules in the condensed phase would likely entail substantial void volume due to imperfect packing; thus the condensed droplets would still be unstable under increased pressure as depicted in Figure 5d. At the same time, because the condensed phase has less solvent-exposed protein surface than the dilute phase—irrespective of whether the condensed phase is consisting of proteins that are completely folded (Figure 5e) or partially unfolded (Figure 5g), TMAO is expected to always stabilize the condensed phase relative to the dilute phase. This general physical picture is illustrated by the schematic drawings in Figures 5f,g showing partially unfolded γ-D-crystallin molecules with their C-terminal domains largely folded, a conformational feature suggested by experiment and simulation for destabilizing conditions.62,63 However, in view of the aforementioned lack of changes in secondary structure in the condensed phase relative to the dilute phase, we consider the alternate scenario with substantial partial unfolding in the condensed phase rather unlikely.



Figure 5. Proposed conceptual framework for the TMAO stabilization of phase-separated γD crystallin condensates. (a-c) Folded structures of human γ-D-crystallin (PDB id:1HK0) depicted by (a) a ribbon diagram (blue: N-terminus, red: C-terminus), and space-filling representations highlighting (b) surface hydrophobicity,64 (green: maximally hydrophobic, red: least hydrophobic/most hydrophilic), and (c) residue types. These drawings are generated by NGL Viewer using its default color codes for hydrophobicity and residue types, e.g., Arg and Asp are colored deep blue and deep red respectively.65 (d, e) Individual folded structures of human γ-D crystallin (residues color-coded as in (c)) are shown with their molecular (solventexcluded) surfaces (JSmol66). Water and TMAO molecules are schematically depicted, respectively, as blue and red circles (sizes not drawn to scale). As in ref. 21, the cyan areas are accessible to some part of a water molecule, whereas white areas are voids. Owing to transient contacts with imperfect packing, the condensed phase of the phase-separated state (left) is expected to harbor more voids (white area) statistically. Hydrostatic pressure favors the homogeneous state because it has less void volume on average and therefore a reduced total volume (d). However, with TMAO, the homogeneous state entails more unfavorable TMAO-protein interactions, giving rise to an opposing thermodynamic driving force toward the phase-separated state (e). Here, the unfavorable nature of TMAO-protein interaction is underscored schematically by a relative lack of TMAO molecules in the direct vicinity of the protein molecules in (e). (f) Schematic of a partially unfolded γ-D-crystallin conformation, wherein the N-terminal domain is disordered (represented as a chain of beads) but the Cterminal domain remains largely folded [depicted in the style for the folded structures in (d, e)]. (g) An alternate scenario in which some or all of the γ-D-crystallin molecules are partially unfolded in the condensed phase. The present measurements on human and rat γ-D-crystallin show LLPS under ambient pressure at 5 oC and 8 oC, respectively. These cloud-point temperatures are well below normal mammalian body temperatures. However, if mutations, amino acid oxidation or deamidation cause the cloud point to rise above body temperature, LLPS of crystallin could occur in the living human lens to result in the formation of cataract. Our data also show that


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pressures as small as several tens of bars can drive the system in the opposite direction across the LLPS boundary to the homogeneous solution phase. These findings are of biological relevance, for example, for understanding the composition of eye lens proteins of artic fishes.45 Clearly, their crystallins must be able to avoid LLPS at the low temperatures of their living environment, but perhaps the elevated hydrostatic pressure in the ocean would help avoid LLPS-induced opacity of their eye lenses. This and other similarly intriguing questions remain to be explored. Biological cells survive extreme environmental conditions in part by using osmolytes to rescue proteins from denaturation and subsequent aggregation. We studied the effect of TMAO, arguably the most prominent osmolyte in deep-sea organisms.50 In recent years, TMAO at concentrations of several hundred mM were shown to increase the temperature and pressure stability of folded proteins and nucleic acids.3,67-71 Here we find further that TMAO also stabilizes certain protein droplets—which can be critical for biological function— against pressure-induced dissolution. Although TMAO is not known to modulate LLPS of crystallin in vivo, the present data on γ-D-crystallin are a good starting point to study the competing effects of pressure and TMAO on biomolecular condensates in general. 4. Conclusions In summary, using human and rat γ-D-crystallin as model systems, we have studied how their LLPS depends on hydrostatic pressure and TMAO. A general understanding of how the properties of biomolecular condensates depend on these environmental variables is important for normal as well as disease-causing processes. This knowledge will shed light on the possible evolutionary advantages of utilizing LLPS for biological function and provide insights into the self-organization principles underpinning cellular evolution in the face of external stresses. Hypothetically, the first steps in the origin of biological cells could have been the phase separation of biomacromolecules into liquid coacervates.7 As the birth place of life on Earth could have been the deep sea,32 studies along the line here of the effect of pressure and low temperatures—as encountered in the ocean depths—on the LLPS process should bear on the possible formation of protocells under prebiotic conditions. The data we reported indicate that upregulation of TMAO can stabilize the droplet phase even at the hundreds-of-bar level of pressures encountered in the deep sea (the average pressure in the world’s oceans is ~400 bar). In a broader technological context, knowledge of the LLPS behavior of proteins as a function of all thermodynamic variables, including pressure, is essential in areas such as protein crystallization (LLPS occurs generally at conditions where the protein is metastable with respect to crystallization), protein purification, formulation development in pharmaceutical applications, and high-pressure food processing.25,72-74 There is no shortage of productive avenues to pursue by building on the effort reported in this work. Acknowledgements R.W. acknowledges funding from Cluster of Excellence RESOLV (EXC-1069), DFG Research Unit FOR 1979, and the Deep Carbon Observatory (Alfred P. Sloan Foundation). H.S.C.’s research effort is supported by the Canadian Institutes of Health Research grants MOP-84281 and PJT-155930, and Natural Sciences and Engineering Research Council of Canada grant RGPIN-2018-04351. Associated Content Supporting Information: Experimental details and additional figures. References (1) Minton, A. P. How can biochemical reactions within cells differ from those in test tubes? J. Cell Sci. 2006, 119, 2863–2869. (2) Zhou, H.-X.; Rivas, G.; Minton, A. P. Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 2008, 37, 375–397.



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Pressure-Sensitive and Osmolyte-Modulated Liquid-Liquid Phase

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