Physicochemical Characterization of Polymer‐Stabilized Coacervate Protocells

Abstract

The bottom‐up construction of cell mimics has produced a range of membrane‐bound protocells that have been endowed with functionality and biochemical processes reminiscent of living systems. The contents of these compartments, however, experience semidilute conditions, whereas macromolecules in the cytosol exist in protein‐rich, crowded environments that affect their physicochemical properties, such as diffusion and catalytic activity. Recently, complex coacervates have emerged as attractive protocellular models because their condensed interiors would be expected to mimic this crowding better. Here we explore some relevant physicochemical properties of a recently developed polymer‐stabilized coacervate system, such as the diffusion of macromolecules in the condensed coacervate phase, relative to in dilute solutions, the buffering capacity of the core, the molecular organization of the polymer membrane, the permeability characteristics of this membrane towards a wide range of compounds, and the behavior of a simple enzymatic reaction. In addition, either the coacervate charge or the cargo charge is engineered to allow the selective loading of protein cargo into the coacervate protocells. Our in‐depth characterization has revealed that these polymer‐stabilized coacervate protocells have many desirable properties, thus making them attractive candidates for the investigation of biochemical processes in stable, controlled, tunable, and increasingly cell‐like environments.

Liquid–Liquid Phase Separation in Crowded Environments

“…The corresponding maximum has been observed experimentally for cell-free gene expression in solution [ 47 ]. It is interesting to note that an enhanced enzyme activity, similar to what is commonly seen in intermediately crowded conditions, has also been proposed to occur for “overcrowded” condensates [ 48 , 49 , 50 ]. Whether this is a consequence of crowding, or has a different origin, is still an open question, which is beyond the scope of this review.…”

Mimicking Cellular Compartmentalization in a Hierarchical Protocell through Spontaneous Spatial Organization

“…38 Membranized coacervates have prolonged stability under aqueous buffer conditions, as well as a semipermeable exterior that allows transmembrane diffusion of small molecule substrates. 39 Chemical homology between the external membrane and inner proto-organelles (embedded in the cytosol-mimetic coacervate) makes this an exciting system for the exploration of more complex cell-mimetic behaviors (as depicted in Scheme 1). This hierarchical protocell affords us control over the spatial organization of enzymes and their reactions, facilitating the study of two main functional consequences of compartmentalization: the creation of favorable microenvironments and the ability to segregate incompatible components.…”

Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions

“…Extant cells facilitate this function through amphiphilic lipids, usually containing proteins within the membranes that they constitute, but different systems such as de novo cellular compartments/organelles based on amphiphilic proteins and minimal entity protocells are molecularly possible in the context of synthetic cellular top‐down, integrative middle‐out and transforming bottom‐up approaches . In view of the molecular requirements for “cellular” systems, the quest for artificial cells and protocells might also benefit from the interesting reengineered systems used in biotechnology, such as cells, permeabilized cells, cell‐free expression systems in vesicular compartments, polymer‐stabilized coacervate protocells and pickering emulsions including protein‐based systems as examples for compartmentalized catalytic systems. From a physicochemical point of view, questions that arise include how a minimal system based on the smallest number of classes of molecules might look and whether or not functional relations of extant cells can be recorrelated.…”