Coacervate


Coacervate is an aqueous phase rich in macromolecules such as synthetic polymers, proteins or nucleic acids. It forms through liquid-liquid phase separation , leading to a dense phase in thermodynamic equilibrium with a dilute phase. The dispersed droplets of dense phase are also called coacervates, micro-coacervates or coacervate droplets.These structures draw a lot of interest because they form spontaneously from aqueous mixtures and provide stable compartmentalization without the need of a membrane.
The term coacervate was coined in 1929 by Dutch chemist Hendrik G. Bungenberg de Jong and Hugo R. Kruyt while studying lyophilic colloidal dispersions. The name is a reference to the clustering of colloidal particles, like bees in a swarm. The concept was later borrowed by Russian biologist Alexander I. Oparin to describe the proteinoid microspheres proposed to be primitive cells on early Earth. Coacervate-like protocells are at the core of the Oparin-Haldane hypothesis.
A reawakening of coacervate research was seen in 2009 due to the recognition of the role of liquid-liquid phase separation in the formation of certain membraneless organelles by American biophysicist Clifford Brangwynne and Tony Hyman. Liquid organelles share features with coacervate droplets and fueled the study of coacervates for biomimicry.

Thermodynamics

Coacervates are a type of lyophilic colloid; that is, the dense phase retains some of the original solvent – generally water – and does not collapse into solid aggregates, rather keeping a liquid property. Coacervates can be characterized as complex or simple based on the driving force for the LLPS: associative or segregative. Associative LLPS is dominated by attractive interactions between macromolecules, and segregative LLPS is driven by the minimization of repulsive interactions.
The thermodynamics of segregative LLPS can be described by a Flory-Huggins polymer mixing model. In ideal polymer solutions, the free-energy of mixing is negative because the mixing entropy is negative and the interaction enthalpies are all taken as equivalent. In non-ideal solutions, ΔmixH can be different than zero, and the process endothermic enough to overcome the entropic term and favor the de-mixed state. Low molecular-weight solutes will hardly reach such non-ideality, whereas for polymeric solutes, with increasing interactions sites N and therefore decreasing entropic contribution, simple coacervation is much more likely.
The phase diagram of the mixture can be predicted by  experimentally determining the two-phase boundary, or binodal curve. In a simplistic theoretical approach, the binodes are the compositions at which the free energy of de-mixing is minimal, across different temperatures. Alternatively, by minimizing the change in free energy of de-mixing in regards to composition, the spinodal curve is defined. The conditions of the mixture in comparison to the two curves defines the phase separation mechanism: nucleation-growth of coacervate droplets and spinodal decomposition.
Associative LLPS is more complex to describe, as both solute polymers are present in the dilute and dense phase. Electrostatic-based complex coacervates are the most common, and in that case the solutes are two polyelectrolytes of opposite charge. The Voorn-Overbeek approach applies the Debye-Hückel approximation to the enthalpic term in the Flory-Huggins model, and considers two polyelectrolytes of the same length and at the same concentration. Complex coacervates are a subset of aqeous two-phase systems, which also include segregatively separated systems in which both phases are enriched in one type of polymer.

Coacervates in biology

Membraneless organelles , also known as biomolecular condensates, are a form of cell compartmentalization. Unlike classic membrane-bound organelles, MLOs are not separated from their surroundings by a lipid bilayer. MLOs are mostly composed of proteins and nucleic acids, held together by weak intermolecular forces.
MLOs are present in the cytoplasm and in the nucleus. They have been shown to serve various functions: they can store and protect cellular material during stress conditions, they participate in gene expression and they are involved in the control of signal transduction.
It is now widely believed that MLOs form through LLPS. This was first proposed after observing that Cajal bodies and P granules show liquid-like properties, and was later confirmed by showing that liquid condensates can be reconstituted from purified protein and RNA in vitro. However, whether MLOs should be referred to as liquids, remains disputable. Even if initially they are liquid-like, over time some of them maturate into solids.
Many proteins participating in the formation of MLO contain so-called intrinsically disordered regions, parts of the polypeptide chain that can adopt multiple secondary structures and form random coils in solution. IDRs can provide interactions responsible for LLPS, but over time conformational changes may lead to the formation of higher ordered structures and solidification of MLOs. Some MLOs serve their biological role as solid particles, but in many cases transformation from liquid to solid results in the formation of pathological aggregates. Examples of both liquid-liquid phase separating and aggregation-prone proteins include FUS, TDP-43 and hnRNPA1. Aggregates of these proteins are associated with neurodegenerative diseases.

History

At the start of the 20th century, scientists had become interested in the stability of colloids, both the dispersions of solid particles and the solutions of polymeric molecules. It was known that salts and temperature could often be used to cause flocculation of a colloid. The German chemist F.W. Tiebackx reported in 1911 that flocculation could also be induced in certain polymer solutions by mixing them together. In particular, he reported the observation of opalescence when equal volumes of acidified 0.5% “washed” gelatine solution, and 2% gum arabic solution were mixed. Tiebackx did not further analyse the nature of the flocs, but it is likely that this was an example of complex coacervation.
Dutch chemist H. G. Bungenberg-de Jong reported in his PhD thesis two types of flocculation in agar solutions: one that leads to a suspensoid state, and one that leads to an emulsoid state. He observed the emulsoid state under the microscope and described small particles that merged into larger particles, most likely a description of coalescing coacervate droplets. Several years later, in 1929, Bungenberg-de Jong published a seminal paper with his PhD advisor, H. R. Kruyt, entitled “Coacervation. Partial miscibility in colloid systems”. In their paper, they give many more examples of colloid systems that flocculate into an emulsoid state, either by varying the temperature, by adding salts, co-solvents or by mixing together two oppositely charged polymer colloids, and illustrate their observations with the first microscope pictures of coacervate droplets. They term this phenomenon coacervation, derived from the prefix co and the Latin word acervus, which relates to the dense liquid droplets. Coacervation is thus loosely translated as ‘to come together in a heap’. Since then, Bungenberg-de Jong and his research group in Leiden published a range of papers on coacervates, including results on self-coacervation, salt effects, interfacial tension, multiphase coacervates and surfactant-based coacervates.
In the meantime, Russian chemist Alexander Oparin, published a pioneering work in which he laid out his protocell theory on the origin of life. In his initial protocell model, Oparin took inspiration from Graham’s description of colloids from 1861 as substances that usually give cloudy solutions and cannot pass through membranes. Oparin linked these properties to the protoplasm, and reasoned that precipitates of colloids form as clots or lumps of mucus or jelly, some of which have structural features that resemble the protoplasm. According to Oparin, protocells could therefore have formed by precipitation of colloids. In his later work, Oparin became more specific about his protocell model. He described the work of Bungenberg-de Jong on coacervates in his book from 1938, and postulated that the first protocells were coacervates.
Other researchers followed, and in the 1930s and 1940s various examples of coacervation were reported, by Bungenberg-de Jong, Oparin, Koets, Bank, Langmuir and others. In the 1950s and 1960s, focus shifted to a theoretical description of the phenomenon of coacervation. Voorn and Overbeek developed the first mean-field theory to describe coacervation. They estimated the total free energy of mixing as a sum of mixing entropy terms and mean-field electrostatic interactions in a Debye-Hückel approximation. Veis and Aranyi suggested to extend this model with an electrostatic aggregation step in which charge-paired symmetrical soluble aggregates are formed, followed by phase separation into liquid droplets.
In the decades after that, until about 2000, the scientific interest in coacervates had faded. Oparin’s theory on the role of coacervates in the origin of life had been replaced by interest in the RNA world hypothesis. Renewed interest in coacervates originated as scientists recognized the relevance and versatility of the interactions that underlie complex coacervation in self-assembly and responsive materials.
Since 2009, coacervates have become linked to membraneless organelles and there has been a renewed interest in coacervates as protocells.

Complex coacervation

Complex coacervation commonly refers to the liquid-liquid phase separation that results when solutions of two oppositely charged macroions are mixed, resulting in the formation of a dense macroion-rich phase, the precursors of which are soluble complexes.
This has widely been found to be involved in the formation of biomolecular condensates, also known as membraneless organelles within cells.