Protein Liquid-liquid Phase Separation Research Articles

A high-throughput method for exploring the parameter space of protein liquid-liquid phase separation

Numerous proteins can undergo liquid-liquid phase separation (LLPS) in various biological processes. However, it remains a challenge to systematically evaluate the LLPS ability of proteins. Here, we develop a high-throughput protein phase separation (HiPPS) profiling method to delineate LLPS ability of proteins, which we term as the LLPS space of proteins. We next apply HiPPS to explore LLPS space of more than 30 different proteins including RNA-binding proteins, chaperones, amyloid proteins, and enzymes. Our results demonstrate that (1) LLPS is a common property of both unstructured and well-folded proteins; (2) LLPS space of protein can be dynamically reshaped by protein-protein interaction, post-translational modification, and genetic mutation; and (3) multi-component co-LLPS mixture decreases the threshold concentration of individual proteins for LLPS. We anticipate that the HiPPS method may effectively characterize LLPS of individual proteins or protein mixtures in vitro , which is valuable for understanding its role in biological processes. • Development of HiPPS profiling method • IDR and well-folded proteins harbor LLPS abilities in vitro • Protein-protein interaction, PTM, and genetic mutation re-shape LLPS space of protein Protein phase separation is highly sensitive to various conditions. Li et al. establish an automated high-throughput profiling method to quantitatively assess the phase separation ability of different proteins and proteins in complex systems. The method may serve as a useful tool to explore underlying mechanisms of protein phase separation.

Coupling between protein condensates and ordered membrane domains facilitate T cell activation

Liquid-liquid phase separation of proteins have been broadly observed in many cellular machineries, but the biological roles of many of these protein condensates remains ambiguous. Condensates of the membrane protein LAT with Grb2+Sos1 have been implicated in T cell activation, possibly by excluding inhibitory phosphatase CD45 through electrostatic repulsion and/or increasing the dwell time of effector protein Sos1 near the plasma membrane (PM). Intriguingly, LAT has been widely shown to reside in ordered PM domains known as lipid rafts.

Effect of cyclization on the liquid-liquid phase separation of pentaglycine

Liquid-liquid phase separation of proteins is a fast growing area of biophysical inquiry due to the role it plays in the formation of membraneless organelles, disease-associated protein aggregation, and disordered protein domains in signaling. Examining these processes in the context of entropy and enthalpy changes is necessary to establish a thermodynamic foundation with which to understand phase transitions. Our previous work probed the phase separation of glycine-rich pentapeptides and found that a loss of conformational entropy upon phase separation is compensated for by a decrease in peptide interaction enthalpy.

Tuning the formation of protein-nucleic acid condensates

Liquid-liquid phase separation (LLPS) is now widely recognized to occur in a broad variety of cellular interactions, particularly inside the nucleus. Here, we have developed a coarse-grain (CG) model for the LLPS of intrinsically-disordered proteins (IDPs) together with nucleic acids (NAs). The model allows us to probe the effects of changing the salt concentration and protein charge patterning on IDP-NA LLPS. We use as our model peptide the disordered C-terminus of histone H1 (CH1), which phase separates with short dsDNA fragments at physiological salt concentrations.

Chemical tools for study and modulation of biomolecular phase transitions.

Biomolecular phase transitions play an important role in organizing cellular processes in space and time. Methods and tools for studying these transitions, and the intrinsically disordered proteins (IDPs) that often drive them, are typically less developed than tools for studying their folded protein counterparts. In this perspective, we assess the current landscape of chemical tools for studying IDPs, with a specific focus on protein liquid-liquid phase separation (LLPS). We highlight methodologies that enable imaging and spectroscopic studies of these systems, including site-specific labeling with small molecules and the diverse range of capabilities offered by inteins and protein semisynthesis. We discuss strategies for introducing post-translational modifications that are central to IDP and LLPS function and regulation. We also investigate the nascent field of noncovalent small-molecule modulators of LLPS. We hope that this review of the state-of-the-art in chemical tools for interrogating IDPs and LLPS, along with an associated perspective on areas of unmet need, can serve as a valuable and timely resource for these rapidly expanding fields of study.

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.

Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Accurate model of liquid–liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties

Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or "membraneless organelles" such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

Phase separation vs aggregation behavior for model disordered proteins

Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence-dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, stickerfraction, and chain length impact the formation of finite-size aggregates, which can preempt macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" predictive criterion for distinguishing when a model protein undergoes macroscopic phase separation vs finite aggregation. Additionally, we find that this order parameter is strongly correlated with the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveal that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results suggest that classical LLPS should be the primary phase transition for disordered proteins when short-ranged attractive interactions dominate and suggest a possible reason behind recent findings of widespread phase separation throughout living cells.

Determining the Phase Separation Characteristics of Plant Proteins.

Eukaryotic cells contain numerous membrane-bound and membraneless organelles that provide spatiotemporal control for diverse biological processes. The liquid-liquid phase separation of proteins has been proposed as the driving force behind the formation of membraneless organelles. Here, we describe a method to determine the phase separation activities of proteins in plants. This basic method includes protocols for an in vivo fluorescence recovery after photobleaching assay in Nicotiana benthamiana using transient expression, an in vitro liquid droplet reconstitution assay using purified recombinant proteins, and an in vivo fluorescence recovery after photobleaching assay in Arabidopsis thaliana using stable transgenic plants. With these assays, the phase separation characteristics of a protein can be determined. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Detection of protein phase separation activities in N. benthamiana Support Protocol: Fluorescence recovery after photobleaching assay Basic Protocol 2: Detection of protein phase separation in vitro Basic Protocol 3: Detection of protein phase separation in stable transgenic Arabidopsis thaliana plants.

Deciphering the Role of π-Interactions in Polyelectrolyte Complexes Using Rationally Designed Peptides.

Electrostatic interactions, and specifically π-interactions play a significant role in the liquid-liquid phase separation of proteins and formation of membraneless organelles/or biological condensates. Sequence patterning of peptides allows creating protein-like structures and controlling the chemistry and interactions of the mimetic molecules. A library of oppositely charged polypeptides was designed and synthesized to investigate the role of π-interactions on phase separation and secondary structures of polyelectrolyte complexes. Phenylalanine was chosen as the π-containing residue and was used together with lysine or glutamic acid in the design of positively or negatively charged sequences. The effect of charge density and also the substitution of fluorine on the phenylalanine ring, known to disrupt π-interactions, were investigated. Characterization analysis using MALDI-TOF mass spectroscopy, H NMR, and circular dichroism (CD) confirmed the molecular structure and chiral pattern of peptide sequences. Despite an alternating sequence of chirality previously shown to promote liquid-liquid phase separation, complexes appeared as solid precipitates, suggesting strong interactions between the sequence pairs. The secondary structures of sequence pairs showed the formation of hydrogen-bonded structures with a β-sheet signal in FTIR spectroscopy. The presence of fluorine decreased hydrogen bonding due to its inhibitory effect on π-interactions. π-interactions resulted in enhanced stability of complexes against salt, and higher critical salt concentrations for complexes with more π-containing amino acids. Furthermore, UV-vis spectroscopy showed that sequences containing π-interactions and increased charge density encapsulated a small charged molecule with π-bonds with high efficiency. These findings highlight the interplay between ionic, hydrophobic, hydrogen bonding, and π-interactions in polyelectrolyte complex formation and enhance our understanding of phase separation phenomena in protein-like structures.

Solubility Parameters of Amino Acids on Liquid-Liquid Phase Separation and Aggregation of Proteins.

The solution properties of amino acids determine the folding, aggregation, and liquid–liquid phase separation (LLPS) behaviors of proteins. Various indices of amino acids, such as solubility, hydropathy, and conformational parameter, describe the behaviors of protein folding and solubility both in vitro and in vivo. However, understanding the propensity of LLPS and aggregation is difficult due to the multiple interactions among different amino acids. Here, the solubilities of aromatic amino acids (SAs) were investigated in solution containing 20 types of amino acids as amino acid solvents. The parameters of SAs in amino acid solvents (PSASs) were varied and dependent on the type of the solvent. Specifically, Tyr and Trp had the highest positive values while Glu and Asp had the lowest. The PSAS values represent soluble and insoluble interactions, which collectively are the driving force underlying the formation of droplets and aggregates. Interestingly, the PSAS of a soluble solvent reflected the affinity between amino acids and aromatic rings, while that of an insoluble solvent reflected the affinity between amino acids and water. These findings suggest that the PSAS can distinguish amino acids that contribute to droplet and aggregate formation, and provide a deeper understanding of LLPS and aggregation of proteins.

Thermodynamic Compensation in Peptides Following Liquid-Liquid Phase Separation.

Liquid-liquid phase separation of proteins often incorporates intrinsically disordered proteins or those with disordered regions. Examining these processes via the entropy change is desirable for establishing a quantitative foundation with which to probe and understand these phase transitions. Of interest is the effect of residue sequence on the entropy of the peptide backbone. In this work we model these systems via all atom simulations of liquid-liquid phase separation of peptides. Systems of supersaturated pentapeptides separate into a peptide-dense liquid droplet phase as well as a dilute (saturated) aqueous phase. An analysis of the change in backbone conformational entropy associated with the phase transition was performed. We examined systems of four different pentapeptides (GGGGG, GGQGG, GGNGG, and GGVGG) in order to explore the effect of sequence variation on the conformational entropy, as well as the effect of side chain variation on the physical characteristics of the droplet phases. We find that the loss of conformational entropy that accompanies aqueous → droplet transitions is more than compensated by a decrease in interaction enthalpy as contributions to the free energy change for the process.

Improved coarse-grained model for studying sequence dependent phase separation of disordered proteins.

We present improvements to the hydropathy scale (HPS) coarse-grained (CG) model for simulating sequence-specific behavior of intrinsically disordered proteins (IDPs), including their liquid-liquid phase separation (LLPS). The previous model based on an atomistic hydropathy scale by Kapcha and Rossky (KR scale) is not able to capture some well-known LLPS trends such as reduced phase separation propensity upon mutations (R-to-K and Y-to-F). Here, we propose to use the Urry hydropathy scale instead, which was derived from the inverse temperature transitions in a model polypeptide with guest residues X. We introduce two free parameters to shift (Δ) and scale (µ) the overall interaction strengths for the new model (HPS-Urry) and use the experimental radius of gyration for a diverse group of IDPs to find their optimal values. Interestingly, many possible (Δ, µ) combinations can be used for typical IDPs, but the phase behavior of a low-complexity (LC) sequence FUS is only well described by one of these models, which highlights the need for a careful validation strategy based on multiple proteins. The CG HPS-Urry model should enable accurate simulations of protein LLPS and provide a microscopically detailed view of molecular interactions.

In silico prediction of in vitro protein liquid-liquid phase separation experiments outcomes with multi-head neural attention.

Proteins able to undergo liquid-liquid phase separation (LLPS) in vivo and in vitro are drawing a lot of interest, due to their functional relevance for cell life. Nevertheless, the proteome-scale experimental screening of these proteins seems unfeasible, because besides being expensive and time-consuming, LLPS is heavily influenced by multiple environmental conditions such as concentration, pH and temperature, thus requiring a combinatorial number of experiments for each protein. To overcome this problem, we propose a neural network model able to predict the LLPS behavior of proteins given specified experimental conditions, effectively predicting the outcome of in vitro experiments. Our model can be used to rapidly screen proteins and experimental conditions searching for LLPS, thus reducing the search space that needs to be covered experimentally. We experimentally validate Droppler's prediction on the TAR DNA-binding protein in different experimental conditions, showing the consistency of its predictions. A python implementation of Droppler is available at https://bitbucket.org/grogdrinker/droppler. Supplementary data are available at Bioinformatics online.

Stress Granule Protein Features Allow for Accurate Prediction of Biological Condensate Localization with GraPES

Biological condensates have been thrust to the forefront of molecular biology over the past decade for their implications in enzymatic activity, neurodegenerative diseases, and cellular organization. It is thought that many of these membraneless organelles are formed through a process known as liquid-liquid phase separation, in which key protein or nucleic acid drivers seed the formation of protein-rich foci within the cellular milieu. Stress granules are stress-induced assemblies that are belong to this group of biological condensates and are of keen interest to the biomolecular research community due to being linked to both long-term cell viability and a variety of protein aggregation-based diseases. Recently, a large amount of proteomic data has been generated that provides unprecedented insight into stress granule composition and stands as fruitful ground for further analysis. Interrogation of this data revealed that stress granule proteins are enriched in features that favor protein liquid-liquid phase separation. Proteins within stress granules were found to be more disordered, soluble, and abundant than their proteome and cytosolic controls while also having an increased potential for post-translational modifications. Furthermore, these “stress granuleomes” were found to be enriched for multivalent character by possessing multiple ordered domains and being likely to interact with RNA maintaining a high level of protein-protein interactions under basal conditions. Our findings are consistent with the notion that stress granule formation is driven by protein liquid-liquid phase separation. Furthermore, stress granule proteins appear poised near solubility limits while possessing the ability to dynamically alter their phase behavior in response to external threat. We culminate results from our analysis into novel predictors for granule incorporation in yeast and mammalian cells that out performs similar computational tools. Using this predictor, we were able to correctly identify new stress granule components, two of which we validated with colocalization microscopy in mammalian tissue culture cells [1]. We then developed a second series of sequence-base predictors and integrate all of these computational tools into a user-friendly web-based interface, called GraPES (Granule Protein Enrichment Server), where users can either look up a variety of pre-calculated likelihood z-scores for human and yeast proteins or obtain novel predictions from their own input FASTA formatted protein sequences.

Taurine suppresses liquid-liquid phase separation of lysozyme protein.

Taurine is a compatible osmolyte that confers stability to proteins. Recent studies have revealed that liquid-liquid phase separation (LLPS) of proteins underlie the formation of membraneless organelles in cells. In the present study, we evaluated the role of taurine on LLPS of hen egg lysozyme. We demonstrated that taurine decreases the turbidity of the polyethylene glycol-induced crowding solution of lysozyme. We also demonstrated that taurine attenuates LLPS-dependent cloudiness of lysozyme solution with 0.5 or 1M NaCl at a critical temperature. Moreover, we observed that taurine inhibits LLPS formation of a heteroprotein mix solution of lysozyme and ovalbumin. These data indicate that taurine can modulate the formation of LLPS of proteins.

Adenosine triphosphate energy-independently controls protein homeostasis with unique structure and diverse mechanisms.

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid-liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2-12 mM, much higher than required for its previously-known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic-acid-binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding-induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π-π, π-cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy-independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein-aggregation diseases.

Aberrant phase separation and cancer.

Eukaryotic cells are intracellularly divided into numerous compartments or organelles, which coordinate specific molecules and biological reactions. Membrane-bound organelles are physically separated by lipid bilayers from the surrounding environment. Biomolecular condensates, also referred to membraneless organelles, are micron-scale cellular compartments that lack membranous enclosures but function to concentrate proteins and RNA molecules, and these are involved in diverse processes. Liquid-liquid phase separation (LLPS) driven by multivalent weak macromolecular interactions is a critical principle for the formation of biomolecular condensates, and a multitude of combinations among multivalent interactions may drive liquid-liquid phase transition (LLPT). Dysregulation of LLPS and LLPT leads to aberrant condensate and amyloid formation, which causes many human diseases, including neurodegeneration and cancer. Here, we describe recent findings regarding abnormal forms of biomolecular condensates and aggregation via aberrant LLPS and LLPT of cancer-related proteins in cancer development driven by mutation and fusion of genes. Moreover, we discuss the regulatory mechanisms by which aberrant LLPS and LLPT occur in cancer and the drug candidates targeting these mechanisms. Further understanding of the molecular events regulating how biomolecular condensates and aggregation form in cancer tissue is critical for the development of therapeutic strategies against tumorigenesis.