Acoustically trapped periodic arrays of horseradish peroxidase (HRP)-loaded poly(diallydimethylammonium chloride) / adenosine 5′-triphosphate coacervate microdroplet-based protocells exhibit a spatiotemporal biochemical response when exposed to a codiffusing mixture of substrate molecules (o-phenylenediamine (o-PD) and hydrogen peroxide (H2O2)) under nonequilibrium conditions. Unidirectional propagation of the chemical concentration gradients gives rise to time- and position-dependent fluorescence signal outputs from individual coacervate microdroplets, indicating that the organized protocell assembly can dynamically sense encoded information in the advancing reaction-diffusion front. The methodology is extended to arrays comprising spatially separated binary populations of HRP- or glucose oxidase-containing coacervate microdroplets to internally generate a H2O2 signal that chemically connects the two protocell communities via a concerted biochemical cascade reaction. Our results provide a step toward establishing a systematic approach to study dynamic interactions between organized protocell consortia and propagating reaction-diffusion gradients, and offer a new methodology for exploring the complexity of protocellular communication networks operating under nonequilibrium conditions.

pigments is of interest to diverse alternative solar energy technologies including photoelectrochemical cells, [4][5][6][7][8][9][10][11][12][13] biosensing, [14,15] photosensing, [16] molecular electronics, [7] and solar fuel synthesis. [17][18][19] Studies have focused in the main on Photosystem I from cyanobacteria [20][21][22] and the RC and RC-LH1 complexes from purple photosynthetic bacteria such as Rhodobacter (Rba.) sphaeroides [23][24][25][26] (Figure 1a,b). This latter organism is a popular source of photoproteins because it is possible to apply extensive protein engineering to its well-characterized RC, enabling high-yield expression and purification of proteins with specifically tailored properties or substantial modifications.A feature of natural photosystems is selective harvesting of certain regions of the solar spectrum, the most obvious illustration being the predominant green color of plant photosynthetic tissues that arises from relatively strong absorbance of red and blue light by chlorophyll and carotenoid pigments. As Rba. sphaeroides synthesizes bacteriochlorophyll (BChl) as its primary photosynthetic pigment its RC exhibits strong absorbance in the near-infrared between 700 and 950 nm, and in the near-UV below 420 nm, but its absorbance across the visible region is relatively weak (Figure 1c). A limitation in the use of this protein in device technologies is therefore suboptimal harvesting of light energy across much of the region where the solar radiation at the earth's surface is maximal, [27] and this limitation is manifest in action spectra of photocurrent density in photoelectrochemical cells based on Rba. sphaeroides pigment-proteins. [28][29][30][31][32][33] In this study we investigated directed self-assembly of conjugates between genetically engineered Rba. sphaeroides RCs and water-soluble cadmium telluride (CdTe) quantum dots (QDs). The tuneable optical properties of these semiconductor nanocrystals have been exploited in a variety of technologies including solar cells and diverse biological applications. [34][35][36] The particular QDs employed in the present work have broad absorbance across the visible spectrum and an emission band centered at 750 nm that overlaps with RC absorbance bands centered at 760 and 800 nm (Figure 1c). These QDs therefore were capable of acting as a synthetic light harvesting system for energy transfer [37,38] and charge separation [23][24][25][26] in the Rba. sphaeroides RC (Figure 1b).Photoreaction centers facilitate the solar energy transduction at the heart of photosynthesis and there is increasing interest in their incorporation into biohybrid devices for solar energy conversion, sensing, and other applications. In this work, the self-assembly of conjugates between engineered bacterial reaction centers (RCs) and quantum dots (QDs) that act as a synthetic light harvesting system is described. The interface between protein and QD is provided by a polyhistidine tag that confers a tight and specific binding and defines the geometry of the interactio...

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyllcontaining bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both kingdoms have been exploited for solar energy conversion, solar fuel synthesis and sensing technologies, but the energy harvesting abilities of these devices are limited by each protein's individual palette of pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of photosystems using naturally diverse but mechanistically complementary components can be achieved in a predictable fashion through the encoding of adaptable, plug-and-play covalent interfaces.

A challenge associated with the utilisation of bioenergetic proteins in new, synthetic energy transducing systems is achieving efficient and predictable self-assembly of individual components, both natural and man-made, into a functioning macromolecular system. Despite progress with water-soluble proteins, the challenge of programming self-assembly of integral membrane proteins into non-native macromolecular architectures remains largely unexplored. In this work it is shown that the assembly of dimers, trimers or tetramers of the naturally monomeric purple bacterial reaction centre can be directed by augmentation with an α-helical peptide that self-associates into extra-membrane coiled-coil bundle. Despite this induced oligomerisation the assembled reaction centres displayed normal spectroscopic properties, implying preserved structural and functional integrity. Mixing of two reaction centres modified with mutually complementary α-helical peptides enabled the assembly of heterodimers in vitro, pointing to a generic strategy for assembling hetero-oligomeric complexes from diverse modified or synthetic components. Addition of two coiled-coil peptides per reaction centre monomer was also tolerated despite the challenge presented to the pigment-protein assembly machinery of introducing multiple self-associating sequences. These findings point to a generalised approach where oligomers or longer range assemblies of multiple light harvesting and/or redox proteins can be constructed in a manner that can be genetically-encoded, enabling the construction of new, designed bioenergetic systems in vivo or in vitro.

Many strategies for meeting mankind's future energy demands through the exploitation of plentiful solar energy have been influenced by the efficient and sustainable processes of natural photosynthesis. A limitation affecting solar energy conversion based on photosynthetic proteins is the selective spectral coverage that is the consequence of their particular natural pigmentation.Here we demonstrate the bottom-up formation of semi-synthetic, polychromatic photosystems in mixtures of the chlorophyll-based LHCII major light harvesting complex from the oxygenic green plant Arabidopsis thaliana, the bacteriochlorophyll-based photochemical reaction centre (RC) from the anoxygenic purple bacterium Rhodobacter sphaeroides and synthetic quantum dots (QDs). Polyhistidine tag adaptation of LHCII and RC enabled predictable self-assembly of LHCII/RC/QD nanoconjugates, the thermodynamics of which could be accurately modelled and parameterised. The tri-component biohybrid photosystems displayed enhanced solar energy conversion via either direct chlorophyll-to-bacteriochlorophyll energy transfer or an indirect pathway enabled by the QD, with an overall energy transfer efficiency comparable to that seen in natural photosystems.