Bistable Functions and Signaling Motifs in Systems Chemistry: Taking the Next Step Toward Synthetic Cells

Abstract

A key challenge in modern chemistry research is to mimic life-like functions using simple molecular networks and the integration of such networks into the first functional artificial cell. Central to this endeavor is the development of signaling elements that can regulate the cell function in time and space by producing entities of code with specific information to induce downstream activity. Such artificial signaling motifs can emerge in nonequilibrium systems, exhibiting complex dynamic behavior like bistability, multistability, oscillations, and chaos. However, the de novo, bottom-up design of such systems remains challenging, primarily because the kinetic characteristics and energy aspects yielding bifurcation have not yet been globally defined. We herein review our recent work that focuses on the design and functional analysis of peptide-based networks, propelled by replication reactions and exhibiting bistable behavior. Furthermore, we rationalize and discuss their exploitation and implementation as variable signaling motifs in homogeneous and heterogeneous environments.The bistable reactions constitute reversible second-order autocatalysis as positive feedback to generate two distinct product distributions at steady state (SS), the low-SS and high-SS. Quantitative analyses reveal that a phase transition from simple reversible equilibration dynamics into bistability takes place when the system is continuously fueled, using a reducing agent, to keep it far from equilibrium. In addition, an extensive set of experimental, theoretical, and simulation studies highlight a defined parameter space where bistability operates.Analogous to the arrangement of protein-based bistable motifs in intracellular signaling pathways, sequential concatenation of the synthetic bistable networks is used for signal processing in homogeneous media. The cascaded network output signals are switched and erased or transduced by manipulating the order of addition of the components, allowing it to reach "on demand" either the low-SS or high-SS. The pre-encoded bistable networks are also useful as a programming tool for the downstream regulation of nanoscale materials properties, bridging together the Systems Chemistry and Nanotechnology fields. In such heterogeneous cascade pathways, the outputs of the bistable network serve as input signals for consecutive nanoparticle formation reaction and growth processes, which-depending on the applied conditions-regulate various features of (Au) nanoparticle shape and assembly.Our work enables the design and production of various signaling apparatus that feature higher complexity than previously observed in chemical networks. Future studies, briefly discussed at the end of the Account, will be directed at the design and analysis of more elaborate functionality, such as bistability under flow conditions, multistability, and oscillations. We propose that a profound understanding of the design principles facilitating the replication-based bistability and related functions bear implications for exploring the origin of protein functionality prior to the highly evolved replication-translation-transcription machinery. The integration of our peptide-based signaling motifs within future synthetic cells seems to be a straightforward development of the two alternating states as memory and switch elements for controlling cell growth and division and even communication among different cells. We furthermore suggest that such systems can be introduced into living cells for various biotechnology applications, such as switches for cell temporal and spatial manipulations.

Figure 1

Bistability in biology and chemistry. (a) A typical bifurcation diagram. Two stable SS solutions are shown in blue and red lines, and an unstable solution is shown in gray. (b) Bistable control of cell division: mitotic substrate phosphorylation by interlinked kinase–phosphatase creating two distinct states, corresponding to interphase and M phase, and a switch function that prevents the cell from flipping back from the M phase to the interphase state. (c) A chemical reaction–diffusion memory device. The image taken shortly after decreasing the light intensity to within the bistable region is identical to a “face” mask (left), while after 1 h the image remained but the continuous lines transformed into dotted lines (right). Panel b Reproduced from ref (20). Available under a CC BY-NC-ND license. Panel c adapted with permission from ref (23). Copyright 2006 Wiley-VCH.

Scheme 1. (a) A Reversible Replication Reaction Templated by the Thiodepsipeptide Coiled Coils. (b) Typical Helical Wheel Presentation of Amino Acids along the Coiled Coils Interaction Interface. (c) Crystal Structure of the Trimeric Thiodepsipeptide Complex; Z18 Marks the Thioester

Figure 2

Bistability arising in thiodepsipeptide replication networks. a) A chemical reaction that invokes bistability. When initiated with high concentrations of the unfolded precursors E and N, the reaction network reaches the Lss, whereas initiation with a high concentration of the folded replicator R leads to a Hss. (b) UPLC traces obtained for the replication reaction at initiation and SS; reactions initiated either with 90 μM E, 90 μM N, and 10 μM R (left) or with 5 μM E, 5 μM N, and 95 μM R (right), leading to Lss and Hss product distributions, respectively. (c) Bistability diagram obtained by plotting K_app as a function of the initial [R]; in all cases, total peptide concentration [E] + [R] = 100 μM. Reproduced from ref (1). Available under a CC BY-NC-ND license. Copyright Gonen Ashkenasy.

Scheme 2. Coupled Redox-Replication Cycles Drive Bistability

Continuous fueling by TCEP drives disulfide reduction in the redox cycle, facilitating reversible processes within the replication cycle. Reproduced from ref (1). Available under a CC BY-NC-ND license. Copyright Gonen Ashkenasy.

Scheme 3. Signal transduction via pathway modifications

The reaction pathway is altered through consecutive additions of the components while keeping the overall mass and energy limits unchanged.

Figure 3

Kinetic simulation of the bistable system. (a) Reaction rate as a function of total template concentration; zero rates correspond to the SSs. (b) A generic bifurcation diagram; SS concentrations vs the total material [R + E]. (c–e) Contour diagrams showing the parameter space for low (light gray), high (dark gray), and bistable (colored) SSs. K_app values are shown for monostable (low or high) SSs, while ΔK_app values are given for the SS differences between the Hss and Lss. DF = denaturation factor. (a, b) Adapted with permission from ref (2). Copyright 2017 Wiley-VCH. (c–e) Reproduced from ref (1). Available under a CC BY-NC-ND license. Copyright Gonen Ashkenasy.

Figure 4

Environmental controls over the bistable network. (a) Bar graph presenting ΔK_app as a function of a specific perturbation in comparison to the “native” case ΔK_app (multiple colors). The x and y axes are not explicitly defined. (b) Experimental bifurcation diagram: K_app values as a function of the coiled-coil propensity. (c) ΔK_app and helicity values obtained under variable conditions. Numbers next to the data points in (a) and (b) depict the experimental conditions shown in this table, where m = total [R + E]/(μM); t = temperature (°C); s = [S]/(μM); g = [GndCl]/(M); r = studied replicator peptide. Reproduced from ref (1). Available under a CC BY-NC-ND license. Copyright Gonen Ashkenasy.

Figure 5

Continuous “switch and erase” functions. K_app values along the Hss-Lss-Hss-Lss (a) and Lss-Hss-Lss-Hss (b) signal processing. Modified with permission from ref (3). Copyright 2024 Cell Press.

Figure 6

Design principles of the cascade reactions: the bistable network SS outputs are used as memory and regulate Au NP formation reaction and growth processes, in turn affecting the NP morphology, shape and assembly. Modified with permission from ref (4). Copyright 2021 Wiley-VCH.

Figure 7

A general scheme describing the coiled coil replication reaction controlled by initiation and inhibition processes in a CSTR, featuring temporal oscillations in product formation. Modified from ref (78). Copyright 2023 American Chemical Society.

Figure 8

Multistable networks. (a) A schematic diagram of two reactions coupled through competition of E₁ and E₂ to react with the resource molecule N. (b,c) Multistationarity diagrams of two nullcline plots obtained for [R₁] and [R₂] replication, presenting three and four stable SS points (green), respectively. Modified with permission from ref (79). Copyright 2020 Wiley-VCH.