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. 相似文献
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. 相似文献
Heterochromatin protein 1α (HP1α) undergoes liquid–liquid phase separation (LLPS) and forms liquid droplets and gels in vitro, properties that also appear to be central to its biological function in heterochromatin compaction and regulation. Here we use solid‐state NMR spectroscopy to track the conformational dynamics of phosphorylated HP1α during its transformation from the liquid to the gel state. Using experiments designed to probe distinct dynamic modes, we identify regions with varying mobilities within HP1α molecules and show that specific serine residues uniquely contribute to gel formation. The addition of chromatin disturbs the gelation process while preserving the conformational dynamics within individual bulk HP1α molecules. Our study provides a glimpse into the dynamic architecture of dense HP1α phases and showcases the potential of solid‐state NMR to detect an elusive biophysical regime of phase separating biomolecules. 相似文献
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. 相似文献
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. 相似文献
Summary: A series of polyethylene (PE) blends consisting of a high density polyethylene (HDPE) and a linear low density polyethylene (LLDPE) with a butene-chain branch density of 77/1000 carbon was prepared at different concentrations. The LLDPE only crystallized below 50 °C, therefore, above 80 °C and below the melting temperature of HDPE, only HDPE crystallized in the PE blends. A specifically designed multi-step experimental procedure based on thermal analysis technique was utilized to monitor the liquid–liquid phase separation (LLPS) of this set of PE blends. The main step was first to quench the system from the homogeneous temperatures and isothermally anneal them at a prescribed temperature higher than the equilibrium melting temperature of the HDPE for the purpose of allowing the phase morphology to develop from LLPS, and then cool the system at constant rate to record the non-isothermal crystallization. The crystallization peak temperature (Tp) was used to character the crystallization rate. Because LLPS results in HDPE-rich domains where the crystallization rates are increased, this technique provided an experimental measure to identify the binodal curve of the LLPS for the system indicated by increased Tp. The result showed that the LLPS boundary of the blend measured by this method was close to that obtained by phase contrast optical microscopy method. Therefore, we considered that the thermal analysis technique based on the non-isothermal crystallization could be effective to investigate the LLPS of PE blends. 相似文献
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. 相似文献
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.相似文献
Biomolecular condensates consisting of proteins and nucleic acids can serve critical biological functions, so that some condensates are referred as membraneless organelles. They can also be disease-causing, if their assembly is misregulated. A major physicochemical basis of the formation of biomolecular condensates is liquid–liquid phase separation (LLPS). In general, LLPS depends on environmental variables, such as temperature and hydrostatic pressure. The effects of pressure on the LLPS of a binary SynGAP/PSD-95 protein system mimicking postsynaptic densities, which are protein assemblies underneath the plasma membrane of excitatory synapses, were investigated. Quite unexpectedly, the model system LLPS is much more sensitive to pressure than the folded states of typical globular proteins. Phase-separated droplets of SynGAP/PSD-95 were found to dissolve into a homogeneous solution already at ten-to-hundred bar levels. The pressure sensitivity of SynGAP/PSD-95 is seen here as a consequence of both pressure-dependent multivalent interaction strength and void volume effects. Considering that organisms in the deep sea are under pressures up to about 1 kbar, this implies that deep-sea organisms have to devise means to counteract this high pressure sensitivity of biomolecular condensates to avoid harm. Intriguingly, these findings may shed light on the biophysical underpinning of pressure-related neurological disorders in terrestrial vertebrates. 相似文献
We experimentally demonstrated that liquid-liquid phase separation (LLPS) of protein aqueous solutions can be induced by isothermal protein oligomerization. This phenomenon is analogous to LLPS induced by the polymerization of small organic molecules in solution. Specifically, using glutaraldehyde for protein cross-linking, we observed the formation of protein-rich liquid droplets for bovine serum albumin and chicken egg lysozyme at 25 degrees C. These droplets evolved into cross-linked protein microspheres. If the aqueous solutions of the protein monomer do not show LLPS at temperatures lower than the oligomerization temperature, protein-rich droplets are not observed. We experimentally linked the formation of these droplets to the increase of LLPS temperature during protein oligomerization. When macroscopic aggregation competes with LLPS, a rationale choice of pH, polyethylene glycol, and salt concentrations can be used to favor LLPS relative to aggregation. Although glutaraldehyde has been extensively used to cross-link protein molecules, to our knowledge, its use in homogeneous aqueous solutions to induce LLPS has not been previously described. This work contributes to the fundamental understanding of both phase transitions of protein solutions and the morphology of protein condensed phases. It also provides guidance for the development of new methods based on mild experimental conditions for the preparation of protein-based materials. 相似文献
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. 相似文献
Summary: The liquid‐liquid phase separation (LLPS) is often coupled with other ordering processes such as crystallization. In a polyolefin blend system, overwhelming changes in crystallization kinetics due to concentration fluctuation caused by spontaneous spinodal LLPS have been observed. Consequently, we are proposing a new mechanism of “fluctuation‐assisted crystallization”. In this process, the usual nucleation barrier could be overcome (or at least partially) by the spontaneous fluctuation growth of LLPS in the spinodal region.
Time‐resolved polarized optical micrographs for poly(ethylene‐co‐hexene) (PEH)/poly(ethylene‐co‐butene) (PEB) = 40:60 isothermally crystallized at 117 °C for 2 min after LLPS at 135 °C for the times shown and the nucleation rates at 117 °C as a function of LLPS time at 135 °C. 相似文献
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. 相似文献
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. 相似文献