Integrating functional synthetic membranes with biological systems

The tools of synthetic biology could allow the reconstitution of the necessary machinery to create synthetic membranes, as well as the intracellular constituents to generate specific functions, enabling the efficient construction of artificial cells. Herein, we describe relevant aspects of the design and preparation of minimal supramolecular architectures that can faithfully mimic or reconstruct the structure and/or function of living cells. Additionally, we report the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers.

HFSP Long-Term Fellow Roberto J. Brea and colleagues
authored on Mon, 28 September 2015

The construction of artificial cells from purely synthetic components provides a novel methodology to reconstruct life’s functions within unnatural materials. Artificial cells have the potential to shed light on biological processes such as gene expression and energy transduction, as well as organize chemical reactions in nanoscale compartments. Therefore, there has been an increasing interest in developing novel strategies for the incorporation and/or integration of biological components in synthetic capsules to facilitate signaling responses, drug delivery, encapsulation and extended expression of biological species. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of highly ordered membrane architectures with defined functionality (Figure 1A). Bottom-up methodologies that assemble artificial cells from synthetic membranes are well suited to a range of applications, such as integrating functionalized vesicles with biological machinery and creating hybrid minimal cells using non-biological chassis. In our first publication, we specifically cover recent fundamental advances that have improved the state-of-the-art self-assembled synthetic cell membranes and have opened exciting potential applications in biosensing, catalysis, and pharmacology. These studies give us a deeper understanding of the nature of living systems that could bring new insights into the origin of cellular life, and provide novel synthetic chassis for advancing synthetic biology.

Figure 1 A) Bottom-up approaches enable the efficient construction of artificial cells. Synthetic biology makes possible the reconstitution of the necessary machinery to create synthetic membranes, as well as the incorporation and/or integration of biological components. B) Spontaneous reconstitution of transmembrane proteins (CcO, MsbA or PMCA) during the non-enzymatic formation of phospholipid vesicles. Protein is initially solubilized with lysophospholipids, which act as the detergent to form micelle-solubilized protein complexes. Addition of the reactive alkyl precursor and subsequent coupling results in the spontaneous generation of the corresponding proteoliposomes.

Integrating synthetic constructs with biological systems has also opened up novel opportunities for exploring fundamental biological properties and processes, such as the effect of macromolecular crowding or the trafficking of protein across endosomal and mitochondrial membranes. In our second publication, we explore the suitability of bioorthogonal coupling reactions for driving the in situ formation of phospholipid membranes and concomitant spontaneous reconstitution of a variety of membrane proteins, with retention of functionality (Figure 1B). Bioorthogonal proteoliposome formation could be a powerful method for studying membrane proteins and/or their incorporation into synthetic cells.

The feasibility of incorporating transmembrane proteins during lipid preparation was initially demonstrated by using the biorthogonal copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction to spontaneously reconstitute active cytochrome c oxidase (CcO) into a bilayer. Additional experiments show that CcO proton pumping remains functional and that the synthetic membranes formed via CuAAC coupling can maintain a proton gradient. Having shown that purified proteins could be spontaneously reconstituted, we also determined the incorporation of membrane proteins overexpressed in bacteria into artificial membranes. MsbA protein showed excellent co-localization with the synthetic membrane, thus indicating that it could be reconstituted successfully into proteoliposomes directly from cell lysate. As a final test, we reconstituted a mammalian plasma membrane calcium ATPase fusion protein (PMCA2-EGFP) into proteoliposomes by native chemical ligation (NCL). Black lipid membrane recording experiments demonstrate that PMCA2 retained its ability to mediate the transport of calcium ions when embedded in synthetic membranes in situ formed through NCL.

We have shown that appropriate design of the molecular components and optimization of the conditions of self-assembly allow the preparation of synthetic cell membranes to be tailored for specific applications. These “hybrid” artificial cells are being actively investigated due to their potential applications in different fields such as chemistry, medicine, pharmacology and materials science.


[1] Towards Self-Assembled Hybrid Artificial Cells: Novel Bottom-Up Approaches to Functional Synthetic Membranes. R. J. Brea, M. D. Hardy, N. K. Devaraj, Chem. Eur. J. 2015, 21, 12564-12570. doi: 10.1002/chem.201501229.

[2] Spontaneous Reconstitution of Functional Transmembrane Proteins During Bioorthogonal Phospholipid Membrane Synthesis. C. M. Cole, R. J. Brea, Y. H. Kim, M. D. Hardy, J. Yang, N. K. Devaraj, Angew. Chem. Int. Ed. 2015, doi: 10.1002/anie.201504339.

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