Engineering transient dynamics of artificial cells by stochastic distribution of enzymes

Here the authors develop a coacervate micromotor that can display autonomous motion as a result of stochastic distribution of propelling units. This stochastic-induced mobility is validated and explained through experiments and theory. Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors

Human peroxiredoxin 3: the shape-shifting peroxidase as a versatile protein tecton

The biological realm contains numerous examples of nano-scale molecules that can self-assemble into a diverse array of architectures, making them attractive building blocks (or tectons) for applications in bionanotechnology. Proteins are one such biological molecule able to assemble into various three-dimensional structures. Exploring the mechanism and conditions in which these protein structures form is not only useful for the understanding of its biological role, but is also a prerequisite for their use in rational materials design. Human peroxiredoxin 3 (HsPrx3) are ubiquitous antioxidant proteins that can form a plethora of protein architectures: from homodimers that reversibly assemble into dodecameric rings (or toroids), and rings that can further associate into protein tubes. This thesis examines the high molecular weight protein tube structure of HsPrx3 (Chapter 2) and its assembly mechanism (Chapter 3). A 2.8 Å crystal structure of HsPrx3 was elucidated for the first time and was displayed as a short tube composed of three rings. This structure, together with a cryo-electron microscopy reconstruction obtained with collaborators, enabled a novel hypothesis for the biological role of these protein tubes as having a self-associating chaperone function. Using native mass spectrometry, protein tube formation was demonstrated to be formed via a non-commutative mechanism. Protein tube formation was also shown to be reversible, increasing the appeal of HsPrx3 proteins as tectons for bionanotechnology. HsPrx3 proteins react with hydrogen peroxide and upon oxidation, the reduced dodecameric rings disassemble into oxidised homodimers. The relationship between this quaternary structural switch and peroxidase activity was investigated (Chapter 4). Point mutations at the dimer-dimer interface were generated, creating an obligate dimer (S75E HsPrx3) and a stabilised toroid (S78C HsPrx3). Intriguingly, the obligate dimer was minimally active, suggesting that the ring structure is important, but not vital, for active site positioning. This raises interesting questions as to the biological function of this redox-induced structural change. On the other hand, the stabilised toroid was crystallised and the 2.4 Å structure provided a detailed understanding of the interactions that stabilise the dimer-dimer interface. S78C HsPrx3 will be a useful tecton as componentry for future applications. Having gained a deeper understanding of HsPrx3 self-assembly, functionalisation of the protein surface with novel chemistries was explored (Chapter 5). An unnatural amino acid, p-azidophenylalanine, was chosen for in vivo incorporation into HsPrx3 via an E. coli expression system. Although, not entirely successful, this marks a promising initial venture at functionalising HsPrx3

The hallmarks of living systems: Towards creating artificial cells

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

Protein nanorings organized by poly(styrene-block-ethylene oxide) self-assembled thin films

This study explores the use of block copolymer self-assembly to organize Lsmα, a protein which forms stable doughnut-shaped heptameric structures. Here, we have explored the idea that 2-D crystalline arrays of protein filaments can be prepared by stacking doughnut shaped Lsmα protein into the poly(ethylene oxide) blocks of a hexagonal microphase-separated polystyrene-b-polyethylene oxide (PS-b-PEO) block copolymer. We were able to demonstrate the coordinated assembly of such a complex hierarchical nanostructure. The key to success was the choice of solvent systems and protein functionalization that achieved sufficient compatibility whilst still promoting assembly. Unambiguous characterisation of these structures is difficult; however AFM and TEM measurements confirmed that the protein was sequestered into the PEO blocks. The use of a protein that assembles into stackable doughnuts offers the possibility of assembling nanoscale optical, magnetic and electronic structures

Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

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 Ni 2+-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

Engineering transient dynamics of artificial cells by stochastic distribution of enzymes

Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors

Physicochemical characterization of polymer-stabilized coacervate protocells

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

Peroxiredoxin is a Versatile Self-Assembling Tecton for Protein Nanotechnology

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