Temperature,Hydrostatic Pressure,and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

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.     

Liquid–liquid crystalline phase separation in biomolecular solutions

Liquid–liquid phase separation (LLPS) and liquid crystalline (LC) ordering are ubiquitous phenomena in nature, in a variety of biomolecular solutions. Here, we review instances in DNA, nanocellulose, and other systems, where they occur together, leading to the formation of liquid–liquid crystalline phase separation (LLCPS), and we highlight analogies, differences, recent advances, and open questions. Remarkably, the intrinsic fluid yet ordered nature of LC, combined with the spatial confinement induced by LLPS, leads to peculiar biomolecular compartments suitable for a broad range of applications, ranging from material science to synthetic biology. We argue that tools from the LC field help to address still unexplained processes such as the onset of phase transitions in intracellular biomolecular condensates.     

Spatiotemporal organization of coacervate microdroplets

Liquid–liquid phase separation (LLPS) has emerged as a new paradigm in the fields of soft matter, colloid chemistry, prebiotic chemistry, and cell biology. As phase separation is a dynamic assembly process, how to spatiotemporally regulate the assembly and disassembly of these micrometre-sized droplets, which are referred as biomolecular condensates in biology is essential for their diverse applications in various disciplines. Herein, we discuss recent advances in the spatiotemporal control of phase separation using different physical tools and external environmental stimuli in bulk solutions and living cells. Specifically, the exploration of phase transition in a compartmentalized protocellular system, which can bridge the gap between synthetic and intracellular LLPS systems, is summarized, and the challenges and future research directions are discussed.     

Suppression of Liquid-Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid–liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.     

Polyampholyte physics: Liquid–liquid phase separation and biological condensates

Here we review the current understanding of molecular interactions that govern liquid–liquid phase separation (LLPS) of biological condensates. The connection between sequence, chain conformation, and phase separation of intrinsically disordered proteins (IDPs) and their model polyampholytes is discussed. In particular, we highlight how the charge pattern influences the conformation and phase behavior of natural IDPs. We then describe recent results from theoretical treatments of polyampholytes implementing random phase approximation, field-theoretic simulations, and transfer matrix theory that show an increase in charge segregation results in an increased tendency to phase separate.     

Coacervate formation studied by explicit solvent coarse-grain molecular dynamics with the Martini model

Complex coacervates are liquid–liquid phase separated systems, typically containing oppositely charged polyelectrolytes. They are widely studied for their functional properties as well as their potential involvement in cellular compartmentalization as biomolecular condensates. Diffusion and partitioning of solutes into a coacervate phase are important to address because their highly dynamic nature is one of their most important functional characteristics in real-world systems, but are difficult to study experimentally or even theoretically without an explicit representation of every molecule in the system. Here, we present an explicit-solvent, molecular dynamics coarse-grain model of complex coacervates, based on the Martini 3.0 force field. We demonstrate the accuracy of the model by reproducing the salt dependent coacervation of poly-lysine and poly-glutamate systems, and show the potential of the model by simulating the partitioning of ions and small nucleotides between the condensate and surrounding solvent phase. Our model paves the way for simulating coacervates and biomolecular condensates in a wide range of conditions, with near-atomic resolution.

Martini 3 force field can capture the experimental trends of complex coacervates and can be extended to gain physical insight on the mechanisms that drive the formation of LLPS.     

Studying phase separation in confinement

Cells organize their interior through membrane-bound organelles and through membraneless condensates that are formed by liquid–liquid phase separation (LLPS). The complex process of coacervation that is involved in LLPS is challenging to study in living cells. Hence, studying coacervation in cell-mimicking synthetic containers can yield valuable insights. Here, we review recent progress with respect to studying LLPS (particularly coacervation) in artificial compartments, from water-in-oil droplets to membranous liposomes. We describe different strategies to form and control coacervates in microconfinements and to study their physicochemical and biological characteristics. We also describe how coacervation can itself be used in container formation. This review highlights the importance of in vitro coacervate studies for understanding cellular biology and for designing synthetic cells.     

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.     

Porphyrin/Ionic-Liquid Co-assembly Polymorphism Controlled by Liquid–Liquid Phase Separation

Understanding and controlling multicomponent co-assembly is of primary importance in different fields, such as materials fabrication, pharmaceutical polymorphism, and supramolecular polymerization, but these aspects have been a long-standing challenge. Herein, we discover that liquid–liquid phase separation (LLPS) into ion-cluster-rich and ion-cluster-poor liquid phases is the first step prior to co-assembly nucleation based on a model system of water-soluble porphyrin and ionic liquids. The LLPS-formed droplets serve as the nucleation precursors, which determine the resulting structures and properties of co-assemblies. Co-assembly polymorphism and tunable supramolecular phase transition behaviors can be achieved by regulating the intermolecular interactions at the LLPS stage. These findings elucidate the key role of LLPS in multicomponent co-assembly evolution and enable it to be an effective strategy to control co-assembly polymorphism as well as supramolecular phase transitions.     

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer''s disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Biomolecular condensates sequester an aggregation-prone peptide and prevent its aggregation, showing that heterotypic interactions within the condensates can prevent the formation of amyloid fibrils, despite the local increase in concentration.     

Porphyrin/Ionic‐Liquid Co‐assembly Polymorphism Controlled by Liquid–Liquid Phase Separation

Understanding and controlling multicomponent co‐assembly is of primary importance in different fields, such as materials fabrication, pharmaceutical polymorphism, and supramolecular polymerization, but these aspects have been a long‐standing challenge. Herein, we discover that liquid–liquid phase separation (LLPS) into ion‐cluster‐rich and ion‐cluster‐poor liquid phases is the first step prior to co‐assembly nucleation based on a model system of water‐soluble porphyrin and ionic liquids. The LLPS‐formed droplets serve as the nucleation precursors, which determine the resulting structures and properties of co‐assemblies. Co‐assembly polymorphism and tunable supramolecular phase transition behaviors can be achieved by regulating the intermolecular interactions at the LLPS stage. These findings elucidate the key role of LLPS in multicomponent co‐assembly evolution and enable it to be an effective strategy to control co‐assembly polymorphism as well as supramolecular phase transitions.     

An Inorganic Biopolymer Polyphosphate Controls Positively Charged Protein Phase Transitions

Polyphosphate (PolyP) is one of the most compact inorganic polyanionic biopolymers that participates in various physiological processes. However, the mechanism of the interaction between polyP and proteins remains poorly understood. Herein, we report that polyP can interact with positively charged green fluorescent protein, +36GFP, resulting in liquid–liquid phase separation (LLPS) by intermolecular electrostatic interactions in cells. Upon nutrient deprivation, genetically engineered Citrobacter freundii accumulates intracellular polyP at a rate of 210 μm min^?1, resulting in the compartmentation of +36GFP at the cell poles within 1 h. Medium chain‐length polyP (60‐mer) could induce the formation of +36GFP coacervates in vitro at a protein concentration as low as 200 nm , which is of the same magnitude as native proteins. In contrast, shorter polyP (14‐mer) could not induce LLPS under the same conditions. This may offer a general approach to manipulate protein–protein interactions through LLPS.     

Determining the Physico-Chemical Composition of Biomolecular Condensates from Spatially-Resolved NMR

Liquid-Liquid phase separation has emerged as fundamental process underlying the formation of biomolecular condensates. Insights into the composition and structure of biomolecular condensates is, however, complicated by their molecular complexity and dynamics. Here, we introduce an improved spatially-resolved NMR experiment that enables quantitative analysis of the physico-chemical composition of multi-component biomolecular condensates in equilibrium and label-free. Application of spatially-resolved NMR to condensates formed by the Alzheimer's disease-associated protein Tau demonstrates decreased water content, exclusion of the molecular crowding agent dextran, presence of a specific chemical environment of the small molecule DSS, and ≈150-fold increased concentration of Tau inside the condensate. The results suggest that spatially-resolved NMR can have a major impact in understanding the composition and physical chemistry of biomolecular condensates.     

Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to elucidate the mechanism of LLPS and the subsequent aggregation process. In this study, we showed that ataxin-3, which is associated with Machado–Joseph disease, exhibits LLPS in an intracellular crowding environment mimicked by biopolymers, and proposed that a single droplet formed in LLPS can be quantified using Raman microscopy in a label-free manner. We succeeded in evaluating the protein concentration and identifying the components present inside and outside a droplet using the O–H stretching band of water as an internal intensity standard. Only water and protein were detected to be present inside droplets with crowding agents remaining outside. The protein concentration in a droplet was dependent on the crowding environment, indicating that the protein concentration and intracellular environment should be considered when investigating LLPS. Raman microscopy has the potential to become a powerful technique for clarifying the chemical nature of LLPS and its relationship with protein aggregation.

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes. We have established a method to quantify a single droplet formed by LLPS using the Raman band of water as an internal standard.     

Comparison between protein-polyethylene glycol (PEG) interactions and the effect of PEG on protein-protein interactions using the liquid-liquid phase transition

Phase transitions of protein aqueous solutions are important for protein crystallization and biomaterials science in general. One source of thermodynamic complexity in protein solutions and their phase transitions is the required presence of additives such as polyethylene glycol (PEG). To investigate the effects of PEG on the thermodynamic behavior of protein solutions, we report measurements on the liquid-liquid phase separation (LLPS) of aqueous bovine serum albumin (BSA) in the presence of relatively small amounts of PEG with an average molecular weight of 1450 g/mol (PEG1450) as a model system. We experimentally characterize two thermodynamically independent properties of the phase boundary: (1) the effect of PEG1450 concentration on the LLPS temperature, (2) BSA/PEG1450 partitioning in the two liquid coexisting phases. We then use a thermodynamic perturbation theory to relate the first property to the effect of PEG concentration on protein-protein interactions and the second property to protein-PEG interactions. As criteria to determine the accuracy of a microscopic model, we examine the model's ability to describe both experimental thermodynamic properties. We believe that the parallel examination of these two properties is a valuable tool for verifying the validity of existing models and for developing more accurate ones. For our system, we have found that a depletion-interaction model satisfactorily explains both protein-PEG interactions and the effect of PEG concentration on protein-protein interactions. Finally, due to the general importance of LLPS, we will experimentally show that protein-PEG-buffer mixtures can exhibit two distinct types of liquid-liquid phase transitions.     

Modern optical microscopy methods to study biomolecular condensates

Cells achieve highly intricate internal organization via membrane-bound and membraneless organelles. Research over the past decade has implicated liquid–liquid phase separation, a phenomenon by which charged and often disordered biological macromolecules assemble into reversible liquid-like condensates, as the mechanism of formation of membraneless organelles in cells. During the same period, optical microscopy saw exciting advancements, including the super-resolution revolution, that were quickly adopted by researchers in the biological community. Today, there exists a vast library of techniques capable of providing unprecedented information regarding the formation, function, and dynamics of biomolecular condensates. In this review, we discuss a select number of modern optical microscopy methods that are particularly suited for studying biomolecular condensates both in vitro and in cells, as well as the associated technical challenges.     

Dynamic Spatial Formation and Distribution of Intrinsically Disordered Protein Droplets in Macromolecularly Crowded Protocells

Elastin‐like polypeptides (ELPs) have been proposed as a simple model of intrinsically disordered proteins (IDPs) which can form membraneless organelles by liquid–liquid phase separation (LLPS) in cells. Herein, the behavior of fluorescently labeled ELP is studied in cytomimetic aqueous two‐phase system (ATPS) encapsulated protocells that are formed using microfluidics, which enabled confinement, changes in temperature, and statistical analysis. The spatial organization of ELP could be observed in the ATPS. Furthermore, changes in temperature triggered the dynamic formation and distribution of ELP‐rich droplets within the ATPS, resulting from changes in conformation. Proteins were encapsulated along with ELP in the synthetic protocells and distinct partitioning properties of these proteins and ELP in the ATPS were observed. Therefore, the ability of ELP to coacervate with temperature can be maintained inside a cell‐mimicking system.     

Photo-Responsive Phase-Separating Fluorescent Molecules for Intracellular Protein Delivery

Cellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo-responsive phase-separating fluorescent molecule ( PPFM ) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid-liquid phase separation ( LLPS ), which disintegrate upon photoirradiation with a 405 nm light-emitting diode ( LED ) lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo-responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload.     

Stable Prenucleation Calcium Carbonate Clusters Define Liquid–Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is an intermediate step during the precipitation of calcium carbonate, and is assumed to play a key role in biomineralization processes. Here, we have developed a model where ion association thermodynamics in homogeneous phases determine the liquid–liquid miscibility gap of the aqueous calcium carbonate system, verified experimentally using potentiometric titrations, and kinetic studies based on stopped‐flow ATR‐FTIR spectroscopy. The proposed mechanism explains the variable solubilities of solid amorphous calcium carbonates, reconciling previously inconsistent literature values. Accounting for liquid–liquid amorphous polymorphism, the model also provides clues to the mechanism of polymorph selection. It is general and should be tested for systems other than calcium carbonate to provide a new perspective on the physical chemistry of LLPS mechanisms based on stable prenucleation clusters rather than un‐/metastable fluctuations in biomineralization, and beyond.