No abstract

Despite the astonishing diversity and complexity of living systems, they all share five common hallmarks: compartmentalization, growth and division, information processing, energy transduction and adaptability. In this review, we give not only examples of how cells satisfy these requirements for life and the ways in which it is possible to emulate these characteristics in engineered platforms, but also the gaps that remain to be bridged. The bottom-up synthesis of life-like systems continues to be driven forward by the advent of new technologies, by the discovery of biological phenomena through their transplantation to experimentally simpler constructs and by providing insights into one of the oldest questions posed by mankind, the origin of life on Earth.

A systemic feature of eukaryotic cells is the spatial organization of functional components through compartmentalization. Developing protocells with compartmentalized synthetic organelles is, therefore, a critical milestone toward emulating one of the core characteristics of cellular life. Here we demonstrate the bottom-up, multistep, noncovalent, assembly of rudimentary subcompartmentalized protocells through the spontaneous encapsulation of semipermeable, polymersome proto-organelles inside cell-sized coacervates. The coacervate microdroplets are membranized using tailor-made terpolymers, to complete the hierarchical self-assembly of protocells, a system that mimics both the condensed cytosol and the structure of a cell membrane. In this way, the spatial organization of enzymes can be finely tuned, leading to an enhancement of functionality. Moreover, incompatible components can be sequestered in the same microenvironments without detrimental effect. The robust stability of the subcompartmentalized coacervate protocells in biocompatible milieu, such as in PBS or cell culture media, makes it a versatile platform to be extended toward studies in vitro, and perhaps, in vivo.

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

The potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology.