Acyl Phosphates as Chemically Fueled Building Blocks for Self-Sustaining Protocells.

Lipids spontaneously assemble into vesicle-forming membranes. Such vesicles serve as compartments for even the simplest living systems. Vesicles have been extensively studied for constructing synthetic cells or as models for protocells-the cells hypothesized to have existed before life. These compartments exist almost always close to equilibrium. Life, however, exists out of equilibrium. In this work, we studied vesicle-based compartments regulated by a non-equilibrium chemical reaction network that converts activating agents. In this way, the compartments require a constant or periodic supply of activating agents to sustain themselves. Specifically, we use activating agents to condense carboxylates and phosphate esters into acyl phosphate-based lipids that form vesicles. These vesicles can only be sustained when condensing agents are present; without them, they decay. We demonstrate that the chemical reaction network can operate on prebiotic activating agents, opening the door to prebiotically plausible, self-sustainable protocells that compete for resources. In future work, such protocells should be endowed with a genotype, e.g., self-replicating RNA structures, to alter the protocell's behavior. Such protocells could enable Darwinian evolution in a prebiotically plausible chemical system.

Reciprocal Coupling in Chemically Fueled Assembly: A Reaction Cycle Regulates Self-Assembly and Vice Versa.

In biology, self-assembly of proteins and energy-consuming reaction cycles are intricately coupled. For example, tubulin is activated and deactivated for assembly by a guanosine triphosphate (GTP)-driven reaction cycle, and the emerging microtubules catalyze this reaction cycle by changing the microenvironment of the activated tubulin. Recently, synthetic analogs of chemically fueled assemblies have emerged, but examples in which assembly and reaction cycles are reciprocally coupled remain rare. In this work, we report a peptide that can be activated and deactivated for self-assembly. The emerging assemblies change the microenvironment of their building blocks, which consequently accelerate the rates of building block deactivation and reactivation. We quantitatively understand the mechanisms at play, and we are thus able to tune the catalysis by molecular design of the peptide precursor.

Active droplets through enzyme-free, dynamic phosphorylation.

Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.

Design rules for reciprocal coupling in chemically fueled assembly.

Biology regulates the function and assembly of proteins through non-equilibrium reaction cycles. Reciprocally, the assembly of proteins can influence the reaction rates of these cycles. Such reciprocal coupling between assembly and reaction cycle is a prerequisite for behavior like dynamic instabilities, treadmilling, pattern formation, and oscillations between morphologies. While assemblies regulated by chemical reaction cycles gained traction, the concept of reciprocal coupling is under-explored. In this work, we provide two molecular design strategies to tweak the degree of reciprocal coupling between the assembly and reaction cycle. The strategies involve spacing the chemically active site away from the assembly or burying it into the assembly. We envision that design strategies facilitate the creation of reciprocally coupled and, by extension, dynamic supramolecular materials in the future.

Active coacervate droplets as a model for membraneless organelles and protocells.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA which remains in its active folded state inside the droplets. Finally, we show that in their pathway towards decay, these droplets break apart in multiple droplet fragments. Emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of membrane-less organelles, and we believe that our work could be powerful as a model to study such organelles.