Cells can control the assembly and disassembly of membraneless organelles by enzymatic processes, but similar control has not been achieved in vitro yet. Here we develop ATP-based coacervate droplets as artificial membraneless organelles that can be fully controlled by two cooperating enzymes. Droplets can be generated within a minute following the addition of phosphoenolpyruvate as a substrate, and they can be dissolved within tens of seconds by adding glucose as the second substrate. We show how the rates of droplet generation and dissolution can be tuned by varying the enzyme and substrate concentrations, and we support our findings with a kinetic model of the underlying enzymatic reaction network. As all steps of the coacervate droplet life cycle, including nucleation, coarsening, and dissolution, occur under the same reaction conditions, the cycle can be repeated multiple times. In addition, by carefully balancing the rates of both enzymatic reactions, our system can be programmed to either form or dissolve droplets at specified times, acting as a chemical timer.

Biochemical processes inside the cell take place in a complex environment that is highly crowded, heterogeneous, and replete with interfaces. The recently recognized importance of biomolecular condensates in cellular organization has added new elements of complexity to our understanding of chemistry in the cell. Many of these condensates are formed by liquid-liquid phase separation (LLPS) and behave like liquid droplets. Such droplet organelles can be reproduced and studied in vitro by using coacervates and have some remarkable features, including regulated assembly, differential partitioning of macromolecules, permeability to small molecules, and a uniquely crowded environment. Here, we review the main principles of biochemical organization in model membraneless compartments. We focus on some promising in vitro coacervate model systems that aptly mimic part of the compartmentalized cellular environment. We address the physicochemical characteristics of these liquid phase separated compartments, and their impact on biomolecular chemistry and assembly. These model systems enable a systematic investigation of the role of spatiotemporal organization of biomolecules in controlling biochemical processes in the cell, and they provide crucial insights for the development of functional artificial organelles and cells.

No abstract

Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level.

Coacervates are a type of liquid–liquid phase separated (LLPS) droplets that can serve as models of membraneless organelles (MLOs) in living cells. Peptide–nucleotide coacervates have been widely used to mimic properties of ribonucleoprotein (RNP) granules, but the thermal stability and the role of base stacking is still poorly understood. Here, we report a systematic investigation of coacervates formed by five different nucleoside triphosphates (NTPs) with poly- l -lysine and poly- l -arginine as a function of temperature. All studied combinations exhibit an upper critical solution temperature (UCST), and a temperature-dependent critical salt concentration, originating from a significant nonelectrostatic contribution to the mixing free energy. Both the enthalpic and entropic parts of this nonelectrostatic interaction decrease in the order G/A/U/C/T, in accordance with nucleobase stacking free energies. Partitioning of two dyes proves that the local hydrophobicity inside the peptide–nucleotide coacervates is different for every nucleoside triphosphate. We derive a simple relation between the temperature and salt concentration at the critical point based on a mean-field model of phase separation. Finally, when different NTPs are mixed with one common oppositely charged peptide, hybrid coacervates were formed, characterized by a single intermediate UCST and critical salt concentration. NTPs with lower critical salt concentrations can remain condensed in mixed coacervates far beyond their original critical salt concentration. Our results show that NTP-based coacervates have a strong temperature sensitivity due to base stacking interactions and that mixing NTPs can significantly influence the stability of condensates and, by extension, their bioavailability.

Betalains are water-soluble natural pigments of increasing importance as antioxidants for pharmaceutical use. Although non-phenolic betalains have lower capacity to scavenge radicals compared to their phenolic analogues, both classes perform well as antioxidants and anti-inflammatory agents in vivo. Here we show that meta-hydroxyphenyl betalain (m-OH-pBeet) and phenylbetalain (pBeet) show higher radical scavenging capacity compared to their N-methyl iminium analogues, in which proton-coupled electron transfer (PCET) from the imine nitrogen atom is precluded. The 1,7-diazaheptamethinium system was found to be essential for the high radical scavenging capacity of betalains and concerted PCET is the most thermodynamically favorable pathway for their one-electron oxidation. The results provide useful insights for the design of nature-derived redox mediators based on the betalain scaffold.

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br>

No abstract