Research Methods for Liquid-Liquid Phase Separation of Biological Macromolecules

Chubin Zhao; Hailin Wang

doi:10.7536/PC230222

Progress in Chemistry ›› 2023, Vol. 35 ›› Issue (10) : 1486-1491 DOI: 10.7536/PC230222

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Research Methods for Liquid-Liquid Phase Separation of Biological Macromolecules

Chubin Zhao ¹^,²
, Hailin Wang ¹^,²^,^*

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Abstract

The liquid-liquid phase separation of biological macromolecules is widely observed in various biological systems, and has become an emerging research focus of life science in recent years. Biological macromolecules are continuously enriched through multivalent interaction. When the molecular concentration reaches the dissolution threshold in solution, they will be precipitated from solution in the form of liquid-liquid phase separation. It is closely related to many important biological processes in cells (such as the formation of membraneless organelles). With the deepening of research on phase separation, its research methods are also developing and improving. Based on the principle and characteristics of phase separation, this paper introduces some commonly used research methods of phase separation. It provides the method basis for the subsequent phase separation research and promotes the further development of phase separation techniques and methods.

Contents

1 Principle and characteristics of liquid-liquid separation

2 Imaging technique for liquid-liquid phase separation

2.1 Optical microimaging

2.2 Single-molecule fluorescence imaging

2.3 Fluorescence correlation spectroscopy

3 Theoretical prediction for liquid-liquid separation

3.1 Phase separation prediction and modeling

3.2 Databases of phase separation related proteins

4 Conclusion and outlook

Graphical abstract

Keywords

liquid-liquid phase separation / optical microscopy / fluorescence correlation spectroscopy / molecular simulation

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Chubin Zhao, Hailin Wang. Research Methods for Liquid-Liquid Phase Separation of Biological Macromolecules. Progress in Chemistry, 2023, 35(10): 1486-1491 DOI:10.7536/PC230222

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1 Principle and characteristics of liquid-liquid phase separation

Classical concepts in polymer science suggest that multivalent molecules naturally assemble into large oligomers or polymers when mixed, and that this assembly inherently reduces the solubility of the molecules due to entropy-driven effects, with phase separation occurring when the molecular concentration reaches the solubility threshold in solution^[1]. For biological macromolecules (such as proteins and nucleic acids), this phase separation exists in the form of droplets, which is called liquid-liquid phase separation (LLPS)^[2]. Liquid-liquid phase separation of biomacromolecules has been observed and is thought to be a potentially important mechanism for the dynamic assembly of membraneless organelles, chromatin, and actin filaments^[3⇓~5]. In recent years, more and more studies have shown that liquid-liquid phase separation is involved in the regulation of many important biological processes, such as stress response, gene expression and signal transduction^[6⇓⇓~9].

Many proteins contain structured interaction domains that can cause phase separation, such as Prolin-rich domains and SH3 domains, which often interact with Short linear interaction motifs in other proteins^[10]^[3]. intrinsically disordered regions (intrinsically disordered regions, IDRs) of proteins are also considered to be one of the major driving forces inducing phase separation^[11,12]. In addition, multivalent interactions of proteins with nucleic acids and nucleic acids themselves can also trigger phase separation^[13]^[14]. The liquid-liquid phase separation phenomenon triggered by this multivalent interaction has unique physical properties. Phase-separated droplets are spherical in morphology, fluid, and fuse with each other after contact, allowing for internal rearrangement and external exchange^[15]. Abnormal phase separation can lead to disease. For example, Tau protein can promote the assembly and stabilization of microtubules in healthy neurons^[16]. In the diseased state, the phase-separated droplets formed by Tau protein lose fluidity and harden into a solid state, which impairs the Tau-microtubule interaction and leads to Alzheimer's disease^[17⇓~19]. Therefore, the study of liquid-liquid phase separation of biomacromolecules is of great physiological and pathological significance.

2 Liquid-liquid phase separation imaging technique

2.1 Optical microscopic imaging

Optical microscopic imaging is one of the main research tools to detect liquid-liquid phase separation in vitro and in vivo. According to the physical characteristics of phase-separated droplets, the spherical structure and fusion phenomenon can be detected intuitively, and the internal rearrangement and external exchange can be observed qualitatively or quantitatively. Depending on whether the sample contains a fluorescent label or not, different problems in phase separation can be studied by bright field microscopic imaging and fluorescence microscopic imaging, respectively.

Bright field microscopic imaging technique is often used for phase separation in vitro reconstruction, which has the advantages of no need to label the sample and little influence on the sample, but it can not confirm the molecular composition and material exchange information of phase separation droplets. When in vitro phase separation occurs, the solution will change from clear to turbid, and droplets similar to oil droplets in water will be observed. Phase separation can be better observed and recorded by using polyethylene glycol or lipid-coated slides^[20]. Surewicz et al. Explored the molecular basis of promoting the maturation of phase-separated droplets by bright-field microscopy combined with turbidity measurement, and discussed the potential influence of droplet environment on protein aggregation^[21]. For phase separation in vivo, phase contrast microscopy can be used to reflect the internal structure of cells^[22].

Fluorescence microscopic imaging technology is widely used in the study of phase separation in vivo and in vitro. It has more equipment to choose from, and can observe the interior of phase separation droplets more clearly. It can clarify the molecular composition and material exchange, and carry out quantitative analysis. Because only the fluorescently labeled protein or nucleic acid is observed, the influence of other organelles, chromatin and proteins in vivo is excluded, which is more conducive to the study of phase separation in vivo^[23]. At the same time, because the fluorescence label may affect the sample, it is very important to select the appropriate fluorescence label for fluorescence microscopy imaging. Kanaan et al. Exposed fluorescently labeled Tau protein to a cytoplasmic environment close to physiological levels for a long time, and observed the formation of phase-separated droplets using fluorescence microscopic imaging^[24]. Rosen et al. Used fluorescence microscopy imaging combined with wide-field fluorescence microscopy to observe that the interaction between different synthetic multivalent macromolecules drives liquid-liquid phase separation, revealing the formation mechanism of phase separation^[3]. Zhuang et al. Found that the supersaturated YTHDF protein usually forms condensations at the periphery or junction of Stress granule (SG) clusters by fluorescence microscopic imaging combined with super-resolution imaging, and may promote the formation of SG by reducing the activation energy barrier and the critical size of SG cluster formation^[25].

Fluorescence redistribution after photobleaching (FRAP) experiments, in which high-intensity laser photobleaching of a selected region is used to observe the process of Fluorescence recovery in that region, have been used to demonstrate that several nucleolar subcompartment proteins are capable of phase separation in vitro, but their most common use is to measure the internal fluidity and external material exchange of phase separation droplets^[26,27]. Alberti et al. Observed that the fluorescence of the selected area recovered in a short time by photobleaching the selected area with a confocal microscope, indicating that the G3BP1 molecules in the same stress granule were rapidly rearranged and exchanged with the cytoplasm and adjacent stress granules^[28].

2.2 Single molecule fluorescence imaging

Single molecule fluorescence imaging is a powerful tool to study the conformation and dynamics of Intrinsically disordered proteins (IDP), which has the advantage of detecting the subfraction distribution and dynamic transformation of macromolecules in the system. The signal intensity of a single fluorophore is very weak, and the effect of fluorescence bleaching requires high sensitivity, low noise, and high speed detection equipment. Selvin et al. Developed Single-molecule fluorescence resonance energy transfer (smFRET) technology by extending the traditional whole FRET technology to the single-molecule level, which makes it possible to observe the conformational changes and movements of a single molecule in real time^[29]. Compared with bulk FRET measurement, smFRET technique can not only observe the heterogeneity between molecules and determine the distribution of multiple conformations, but also observe some transiently existing intermediate States as well as unstable States and measure the kinetic parameters of biochemical reactions in real time.

smFRET is a single-molecule measurement technique. When the emission spectrum of a fluorescent donor molecule overlaps with the excitation spectrum of another fluorescent acceptor molecule and their spatial distance is less than 10 nm, the excitation energy of the donor molecule can excite the acceptor molecule to emit fluorescence, while the fluorescence intensity of its own emission decreases^[30]. smFRET can be used to explain conformational changes of proteins during phase separation^[31]. Wu et al. Used smFRET to study the intramolecular conformational changes and intermolecular interactions during the phase separation of Tau protein, and found that the N-terminal and C-terminal domains of Tau protein were opened in the phase separation state, exposing the microtubule-binding region in the middle, which promoted the intermolecular interactions of Tau protein and formed nanoscale oligomers^[32]. Kriwacki et al. Found that nucleolin nucleochrome (NPM1) controls the switching between different phase separation mechanisms through electrostatically driven salt-dependent conformational compression or expansion by smFRET and other methods^[33].

2.3 Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a correlation analysis method for statistical detection of Fluorescence intensity fluctuations of fluorescent molecules in microdomains (Fluorescence correlation spectroscopy) with high sensitivity and high temporal and spatial resolution. It can be used to study the conformational structure and molecular dynamics of biomolecules, and to detect parameters such as local concentration, dissociation constant and diffusion coefficient in phase separation. The classical FCS measurement system is based on a confocal microscope and an excitation laser. The laser is focused into a diffraction-limited spot, which defines a small detection volume with a confocal detection aperture, thus effectively exciting and detecting fluorescence^[34]. When a sufficiently low concentration (typically pmol/L to nmol/L) of fluorophore or fluorescently labeled target molecule solution is placed in the FCS measurement system, unimolecular Brownian diffusion passes through the detection microdomain, resulting in a short burst of detectable fluorescence signal^[35].

Fluorescence correlation spectroscopy has long been used to study phase separation processes in lipid bilayers^[36,37]. In recent years, FCS has been applied to in vivo or in vitro studies of liquid-liquid phase separation of biomacromolecules, exploring the molecular properties inside, outside, and on the boundary of phase-separated droplets^[38]. Using light-induced oligomers to regulate phase separation at specific sites in living cells, Brangwynne et al. Constructed an in vivo phase separation map based on the ability of FCS to quantify the concentration of biomolecules in a single living cell^[39]. King et al. Used FCS to measure the diffusion of single-stranded DNA inside and outside the phase separation droplet in vitro, and studied the phase separation induced by multivalent interactions between single-stranded or double-stranded DNA and cationic peptides, and found that the dominant factor promoting phase separation was the flexible DNA sequence, not the DNA charge distribution^[40]. Enderlein et al. Obtained the first accurately measured absolute value of the diffusion coefficient of the fluorescent dye Cy5 by introducing Dual-focus fluorescence correlation spectroscopy (2fFCS) to simultaneously probe the diffusion kinetics at two separate positions^[41]. Li et al. Developed a simple but powerful quantitative tool, Dual-color fluorescence cross-correlation spectroscopy, for small-scale free-diffusing condensates that cannot be captured by common methods.The concentration and diffusion characteristics of two different fluorescent species and their reaction products in solution were simultaneously quantified, and the formation of nanoscale aggregates beyond the detection limit of conventional fluorescence microscopy was captured, and the size, growth rate, molecular composition of aggregates and binding affinity of recruited molecules were quantified by this technique^[42].

3 Theoretical prediction of liquid-liquid phase separation

3.1 Prediction and simulation of phase separation

Protein liquid-liquid phase separation prediction and simulation is a powerful tool for understanding the mechanism of protein folding and the function of folded proteins, which can provide theoretical predictions for experimental studies and help to explain the molecular mechanism of proteins^[43]. For computational simulation technology, due to the limitation of computer computing power, it is necessary to make a balance between the accuracy of simulation and the efficiency of simulation. The all-atom simulation method with high accuracy and the coarse-grained simulation method with multiple atoms simplified into a single particle to improve the simulation efficiency are commonly used in the prediction and simulation of protein phase separation.

All-atom simulation can provide detailed features of protein sequences and inter-residue interactions consistent with experimental measurements, and has been applied to the study of conformational properties of Tau protein, but it is still limited to smaller systems^[44]^[19]. In order to describe large-scale phenomena, such as the formation of phase-separated droplets, a coarse-grained model is needed. Coarse-grained models can be built by reducing multiple residues to a single ball, a single residue to a single ball, or a single residue to multiple balls, or they can be derived from more detailed atomistic models^[45]^[46]. In order to study the formation mechanism of ribonucleoprotein (RNP) in vivo, Brangwynne et al. Combined in vivo and in vitro studies, simplified each molecule to only a few interaction sites, and performed coarse-grained simulations on a cubic lattice. The results showed that the weak preferential interaction between different components of the droplet could induce the distant spatial structure of different components^[47].

3.2 Phase-Separation-Associated Protein Database

Liquid-liquid phase separation of biomacromolecules is a hot research topic in the field of life science, and several protein databases related to phase separation have been established. The most important databases include PhaSepDB, PhaSePro, LLPSDB v2.0 and DrLLPS.

PhaSepDB (http://db.phasep.pro) is a manually curated database of phase separation-associated proteins, currently updated to PhaSepDB2.1^[48]^[49]. As a relatively large database, PhaSepDB contains 1419 phase-separation-associated protein entries, 770 low-throughput and 7303 high-throughput membraneless organelle-associated protein entries (as of February 2023). The database records the basic information, function and membraneless organelle of each protein, provides the phase separation sequence pattern of all human proteins, and has detailed phase separation protein annotations, including phase separation verification experiments, phase diagrams under different experimental conditions, droplet States, and phase separation regulation information. However, the database does not distinguish between phase separation drivers and regulators, and most phase separation proteins are not supported by reliable evidence.

PhaSepPro (https://phasepro.elte.hu) is a small but sophisticated phase separation protein database containing 121 phase separation-associated proteins validated to drive phase separation (as of February 2023)^[50]. PhaSepPro has more stringent screening criteria and is based entirely on the experimental validation case of phase separation, providing a comprehensive list of phase-separated kinesins and detailed notes on the biological relevance and regulation of phase-separated droplets.

llpsdb v2.0 (http://bio-comp.org.cn/llpsdbv2) is a new version of the database developed based on LLPSDB (http://bio-comp.org.cn/LLPSDB), which contains 2917 phase-separation-related protein entries (as of February 2023)^[51]^[52]. LLPSDB v2.0 collates all proteins and their experimental conditions involved in phase separation in vitro experiments, and records the structural conformation of proteins and the nucleic acid information used in the experiments. The validation experiments provided by this database are mainly focused on in vitro experiments, so they may not be applicable to the in vivo environment.

DrLLPS (http://llps.biocuckoo.cn) is a comprehensive phase separation protein database containing 437887 known and computationally detected phase separation-associated proteins in 164 eukaryotic species (as of February 2023)^[53]. DrLLPS distinguishes scaffold proteins that drive phase separation, regulatory proteins that help regulate phase separation, and potential client proteins that may be involved in the formation of membraneless organelles, with detailed annotations covering 16 aspects of protein intrinsic disorder regions, domains, post-translational modifications, and genetic variation. However, there is no reliable experimental evidence for most phase-separated proteins in this database.

4 Conclusion and prospect

Biomacromolecular liquid-liquid phase separation is a ubiquitous mechanism of cell organization, driven by the liquid nature of intracellular components and macromolecular interactions. Although liquid-liquid phase separation regulates a variety of cellular functions, the specific mechanisms and drivers of its formation are still unclear. At present, the research methods of phase separation mainly include in vitro recombination and in situ imaging. Due to the limitations of each technique, the study of phase separation often requires a combination of technical methods to be validated both in vivo and in vitro. Computational techniques such as molecular modeling can help fill some of the gaps in theoretical, in vivo, and in vitro data, but still fall short. The development of more advanced methods for the study of phase separation is one of the main challenges in this field. In recent years, many new techniques have been applied to the study of phase separation phenomena, such as cryo-electron microscopy, atomic force microscopy and X-ray photon correlation spectroscopy. Although the methods are not yet mature, they have broad application prospects^[54]^[55]^[56]. Recently, Savitski et al. Used cell in situ cryo-electron microscopy to study the three-dimensional reconstruction of mumps virus replisome, and found that the persistent viral replication factory is a dynamic aggregate, and the disordered viral phosphoprotein is an important factor driving its assembly^[57]. Hummer et al. Used fluorescence lifetime imaging of fluore scence resonance energy transfer (FLIM-FRET) technology, combined with coarse-grained molecular simulation, to study the transport channel established by FG-NUP intrinsically disordered proteins in the nuclear pore complex (NPC), and determined that the unknown molecular environment in the nanoscale transport channel could make the FG domain adopt an extended conformation, thus controlling the material between the nucleus and the cytoplasm^[58]. The development of current technology and new methods is very important to explore the phenomenon of phase separation, such as further developing the application of high-resolution cryo-electron microscopy in the study of phase separation protein structure, and developing artificial intelligence, machine learning and other technologies to improve the efficiency and accuracy of phase separation protein prediction and modeling.

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