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Microfluidic characterization of macromolecular liquid-liquid phase separation.

ABSTRACT: Liquid-liquid phase separation plays important roles in the compartmentalization of cells. Developing an understanding of how phase separation is encoded in biomacromolecules requires quantitative mapping of their phase behavior. Given that such experiments require large quantities of the biomolecule of interest, these efforts have been lagging behind the recent breadth of biological insights. Herein, we present a microfluidic phase chip that enables the measurement of saturation concentrations over at least three orders of magnitude for a broad spectrum of biomolecules and solution conditions. The phase chip consists of five units, each made of twenty individual sample chambers to allow the measurement of five sample conditions simultaneously. The analytes are slowly concentrated via evaporation of water, which is replaced by oil, until the sample undergoes phase separation into a dilute and dense phase. We show that the phase chip lowers the required sample quantity by 98% while offering six-fold better statistics in comparison to standard manual experiments that involve centrifugal separation of dilute and dense phases. We further show that the saturation concentrations measured in chips are in agreement with previously reported data for a variety of biomolecules. Concomitantly, time-dependent changes of the dense phase morphology and potential off-pathway processes, including aggregation, can be monitored microscopically. In summary, the phase chip is suited to exploring sequence-to-binodal relationships by enabling the determination of a large number of saturation concentrations at low protein cost.

SUBMITTER: Bremer A

PROVIDER: S-EPMC7658026 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation.

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| S-EPMC6765290 | biostudies-literature

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Project description:A simple method is described for the calculation of two- and three-dimensional phase diagrams describing stability and coexistence curves or surfaces separating one- and two-phase regions in composition/temperature space of a solution containing solute species 1 and 2. The calculation requires a quantitative description of the intermolecular potentials of mean force acting between like (1-1 and 2-2) and unlike (1-2) species. Example calculations are carried out for solutions of species interacting via spherically symmetric square-well potentials as first-order models for protein-protein interaction. When the interaction between species 1 and 2 is more repulsive than those acting between like species, the two-phase region is characterized by an equilibrium between a phase enriched in 1 and depleted in 2 and a phase enriched in 2 and depleted in 1. When the interaction between species 1 and 2 is more attractive than those acting between like species, the two-phase region is characterized by an equilibrium between a phase enriched in both species and a phase depleted in both species. The latter example provides a first-order description of coacervate formation without postulating specific interactions between the two solute species.

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Characterizing protein kinase A (PKA) subunits as macromolecular regulators of PKA RIα liquid-liquid phase separation.

Project description: Not available

| S-EPMC8189721 | biostudies-literature

Bacterial Heterochromatin Formation Through Liquid-Liquid Phase Separation

Project description:Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that silences gene expression. One nucleoid-associated silencing factor is Hfq, which is strongly enriched at prophages and mobile genetic elements. Here we demonstrate that polyphosphate, an ancient and highly conserved polyanion, is essential for the specific binding of Hfq to these regions. Lack of either polyphosphate or Hfq causes unsolicited prophage and transposon mobilization, which drastically increases mutagenesis. We discovered that Hfq and polyphosphate form high molecular weight complexes that interact with DNA in phase-separated viscous condensates. We propose that polyP provides a scaffold that promotes Hfq binding to DNA regions with high intrinsic curvature, and thus serves as one hitherto unknown driver of heterochromatin formation in bacteria.

2021-12-23 | GSE174575 | GEO

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Project description:Liquid-liquid phase separation (LLPS) within the cell can form biological condensates, which are increasingly recognized to play important roles in various biological processes. Most proteins involved in LLPS are known to be intrinsically disordered proteins containing intrinsically disordered regions (IDRs) with low complexity regions (LCRs). The proteins driving LLPS were selected from databases of LLPS-related proteins and then classified into three classes according to the components in the condensates. Through in silico analyses, we found that proteins in the homo class, those that induce LLPS without partner molecules, have different IDRs and LCRs compared with the reference proteome. In contrast, proteins in the other classes, those that induce LLPS with partner proteins (the hetero class) or nucleic acids (the mixed class), did not show a clear difference to the reference proteome in IDRs and LCRs. The hetero-class proteins contained structural domains to serve protein-protein interactions, and the mixed-class ones had the structural domains associated with nucleic acids. These results suggest that IDRs in the homo-class proteins have unique IDRs, which provide multivalent interaction sites required for LLPS, whereas the hetero- and mixed-class proteins can induce LLPS through the combination of the interaction among LCRs, structural domains and nucleic acids.

| S-EPMC10064836 | biostudies-literature

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

Project description: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.

| S-EPMC8674230 | biostudies-literature

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