Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Mechanism of Phase Separation in Aqueous Two-Phase Systems.

Liquid-liquid phase separation underlies the formation of membrane-less organelles inside living cells. The mechanism of this process can be examined using simple aqueous mixtures of two or more solutes, which are able to phase separate at specific concentration thresholds. This work presents the first experimental evidence that mesoscopic changes precede visually detected macroscopic phase separation in aqueous mixtures of two polymers and a single polymer and salt. Dynamic light scattering (DLS) analysis indicates the formation of mesoscopic polymer agglomerates in these systems. These agglomerates increase in size with increasing polymer concentrations prior to visual phase separation. Such mesoscopic changes are paralleled by changes in water structure as evidenced by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis of OH-stretch bands. Through OH-stretch band analysis, we obtain quantitative estimates of the relative fractions of four subpopulations of water structures coexisting in aqueous solutions. These estimates indicate that abrupt changes in hydrogen bond arrangement take place at concentrations below the threshold of macroscopic phase separation. We used these experimental observations to develop a model of phase separation in aqueous media.

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

Current methods for studying intracellular liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) is a ubiquitous process that drives the formation of membrane-less intracellular compartments. This compartmentalization contains vastly different protein/RNA/macromolecule concentrations compared to the surrounding cytosol despite the absence of a lipid boundary. Because of this, LLPS is important for many cellular signaling processes and may play a role in their dysregulation. This chapter highlights recent advances in the understanding of intracellular phase transitions along with current methods used to identify LLPS in vitro and model LLPS in situ.

Hydrogen Bond Arrangement Is Shown to Differ in Coexisting Phases of Aqueous Two-Phase Systems.

Analysis by attenuated total reflection-Fourier transform infrared spectroscopy shows that each coexisting phase in aqueous two-phase systems has a different arrangement of hydrogen bonds. Specific arrangements vary for systems formed by different solutes. The hydrogen bond arrangement is shown to correlate with differences in hydrophobic and electrostatic properties of the different phases of five specific systems, four formed by two polymers and one by a single polymer and salt. The results presented here suggest that the arrangement of hydrogen bonds may be an important factor in phase separation.

The C-Terminus of Perilipin 3 Shows Distinct Lipid Binding at Phospholipid-Oil-Aqueous Interfaces.

Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles.

Skeletal muscle thermogenesis induction by exposure to predator odor.

Non-shivering thermogenesis can promote negative energy balance and weight loss. In this study, we identified a contextual stimulus that induces rapid and robust thermogenesis in skeletal muscle. Rats exposed to the odor of a natural predator (ferret) showed elevated skeletal muscle temperatures detectable as quickly as 2 min after exposure, reaching maximum thermogenesis of >1.5°C at 10-15 min. Mice exhibited a similar thermogenic response to the same odor. Ferret odor induced a significantly larger and qualitatively different response from that of novel or aversive odors, fox odor or moderate restraint stress. Exposure to predator odor increased energy expenditure, and both the thermogenic and energetic effects persisted when physical activity levels were controlled. Predator odor-induced muscle thermogenesis is subject to associative learning as exposure to a conditioned stimulus provoked a rise in muscle temperature in the absence of the odor. The ability of predator odor to induce thermogenesis is predominantly controlled by sympathetic nervous system activation of ß-adrenergic receptors, as unilateral sympathetic lumbar denervation and a peripherally acting ß-adrenergic antagonist significantly inhibited predator odor-induced muscle thermogenesis. The potential survival value of predator odor-induced changes in muscle physiology is reflected in an enhanced resistance to running fatigue. Lastly, predator odor-induced muscle thermogenesis imparts a meaningful impact on energy expenditure as daily predator odor exposure significantly enhanced weight loss with mild calorie restriction. This evidence signifies contextually provoked, centrally mediated muscle thermogenesis that meaningfully impacts energy balance.

Interfacial tension and mechanism of liquid-liquid phase separation in aqueous media.

The organization of multiple subcellular compartments is controlled by liquid-liquid phase separation. Phase separation of this type occurs with the emergence of interfacial tension. Aqueous two-phase systems formed by two non-ionic polymers can be used to separate and analyze biological macromolecules, cells and viruses. Phase separation in these systems may serve as the simple model of phase separation in cells also occurring in aqueous media. To better understand liquid-liquid phase separation mechanisms, interfacial tension was measured in aqueous two-phase systems formed by dextran and polyethylene glycol and by polyethylene glycol and sodium sulfate in the presence of different additives. Interfacial tension values depend on differences between the solvent properties of the coexisting phases, estimated experimentally by parameters representing dipole-dipole, ion-dipole, ion-ion, and hydrogen bonding interactions. Based on both current and literature data, we propose a mechanism for phase separation in aqueous two-phase systems. This mechanism is based on the fundamental role of intermolecular forces. Although it remains to be confirmed, it is possible that these may underlie all liquid-liquid phase separation processes in biology.