Orientation and Incorporation of Photosystem I in Bioelectronics Devices Enabled by Phage Display

Abstract

Interfacing proteins with electrode surfaces is important for the field of bioelectronics. Here, a general concept based on phage display is presented to evolve small peptide binders for immobilizing and orienting large protein complexes on semiconducting substrates. Employing this method, photosystem I is incorporated into solid-state biophotovoltaic cells. Due to the high internal efficiency of the multiprotein complex photosystem I (PSI) to perform light harvesting and charge separation, this key component of photosynthesis potentially enables biophotovoltaic devices in which the active components are biologically grown rather than chemically synthesized. However, owing to the size and structural complexity it remains a challenge to assemble well-oriented monolayers of PSI on surfaces, which is critical for device functionality due to the directional bias of the electron-transfer process. We applied phage-display techniques to identify short binding peptides that afford near-perfect control over the orientation of PSI in self-assembled monolayers (SAMs) once these peptides are functionalized with a surface anchoring group. Almost 100% orientation of PSI on a semiconductor substrate was achieved. We characterized the orientation in junctions by using soft, conformal top contacts comprising eutectic gallium-indium (EGaIn), and using single-complex conducting atomic force microscopy (c-AFM), and demonstrated functionality by fabricating solid-state bulk heterojunction (BJH) solar cells. Phage display (PD) is a very powerful method for the selection of peptides that bind a wide variety of molecules and substrates.1 Initially, it was employed to identify binders against proteins for the development of therapeutic antibodies.2, 3 Closely related applications are the selection of small-molecule enzyme inhibitors from phage-displayed combinatorial peptide libraries4, 5 and the generation of peptide-dendrimer hybrids targeting the structural protein collagen.6 Substrates other than proteins rapidly came into the purview of PD. Small peptide binders were evolved against natural polymers such as chitin,7 synthetic macromolecules including isotactic poly(methyl methacrylate),8 and the conjugated fluorescent polymer poly(p-phenylene vinylene).9 PD even enabled the identification of binders of small organic molecules, ranging from fluorescent dyes10 to volatile explosives such as trinitrotoluene.11 Besides biomolecular substrates, organic small molecule, and macromolecule targets, PD has been used to find binders against inorganic materials, for example, different allotropes of carbon including graphene12 and carbon nanotubes,13 metals such as silver,14 and semiconductors such as GaAs15 or ZnS.16 This utilization of the binding motifs against inorganic substrates allows the fabrication of complex multicomponent devices by displaying selected peptides on the surfaces of phages. For example, viruses with a motif binding single walled carbon nanotubes and a sequence recognizing iron phosphate act as environmentally benign low-temperature biological template for the fabrication of state-of-the-art lithium-ion batteries.17 Virus particles with PD-derived peptides can also act as a biological scaffold for the assembly of active materials for use in dye-sensitized solar cells.18 A natural key building block for transforming light into chemical energy is the multiprotein complex PSI from thermophilic cyanobacteria such as Thermosynechococcus elongatus, which has found widespread use as a model system for structural and function studies. PSI from T. elongatus consists of main subunits shown in Figure 1A,B: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK, PsaL, PsaM, and PsaX.19 Dispersed within the protein scaffold are 127 cofactors: three iron-sulfur (Fe4S4) clusters, one calcium ion, 22 β-carotene, 96 chlorophyll a, 1,2-dipalmitoyl-phosphatidyl-glycerol, one 1,2-distearol-monogalactosyl-diglyceride, and two phylloquinone molecules. Under certain conditions, PSI complexes trimerize with a threefold rotation symmetry axis (C3) oriented perpendicular to the membrane (along with photogenerated charges also flow). The PsaL subunit connects the PSI monomers within the trimer configuration. PsaA and PsaB are the largest subunits containing eleven transmembrane helices. Both subunits bind a large number of chlorophyll molecules and coordinate the electron transport chain (ETC), which is lining the inside of the protein complex. The subunits PsaC, PsaD, and PsaE form a crescent shape and are located at the stroma site of the thylakoid membrane. PsaC coordinates the two terminal Fe4S4 clusters FA and FB of the ETC. Photons harvested by chlorophyll molecules are transferred to the reaction center P700, where charge separation takes place. At P700, the special pair of chlorophylls forms an excited state with a 1.3 V potential difference. From there, the electron is transferred via the primary electron acceptors A0 (Chla), A1 (phylloquinone), FX, FA, and FB (Fe4S4 clusters) within the complex to the external ferredoxin charge carrier. The corresponding positive charge generated at the center of P700+• remains at the opposite side of the protein complex (the lumen side of the thylakoid membrane) and is recharged by electrons from cytochrome c6 (and plastocyanin). The process of light harvesting and charge separation is very efficient, with an internal quantum efficiency close to unity. For that reason, PSI complexes have been incorporated into bio-photonic devices, biosolar and biofuel cells.20-23 Further improvements serving the purpose of photocurrent generation were achieved by implanting Pt nanocrystals in PSI and the fabrication of a nanocomposite by electropolymerization of thioaniline on electrode surfaces.24 However, the application in such devices remains limited by the confines of device architectures, where the principle challenge is overcoming the tendency of PSI to self-assemble with random orientations on surfaces due to its structural complexity—hydrophobic sides, polar stoma, and lumen areas—as well as its relative large size (on the order of 10 nm). To solve this problem, we propose the utilization of PD technology to identify new, short binding peptides capable of directing the orientation of PSI complexes on surfaces. In this Communication, we demonstrate a new phage display protocol to select small peptide binders against PSI trimers chemically bound to the surface of a solid substrate. From this selection procedure, we identified three linear 12 mer peptides with high affinity against the stromal side of PSI. After chemical synthesis and the introduction of a surface anchor, these peptides can bias the orientation of PSI in SAMs such that the electron accepting FB cluster points directing into the substrate; one peptide produces SAMs with nearly 100% orientation. We determined the orientation by c-AFM on single complexes and using EGaIn on the macroscale; the orientation of PSI with respect to the surface of a bottom electrode gives rise to direction-dependent asymmetry in tunneling currents. We demonstrated the functionality of oriented SAMs of PSI by assembling them on indium tin oxide (ITO) electrodes in BJHs biophotovoltaic solar cells where the magnitude of the open-circuit voltage (Voc) correlated to the degree of orientation. Photosystem I from T. elongatus was purified as a trimer and attached to an epoxide-modified surface (see Supporting Information), which resulted in functionalization of amino groups to cause PSI to orient randomly. Next, a commercial library of filamentous bacteriophage M13 expressing 12-amino acid long random-peptide sequences in fusion with their p3 protein (NEB #E8110S) was panned against the immobilized PSI trimer targets. Although the protocol of the vendor was followed, after three rounds of panning and amplification, no selection was achieved. Instead, wild-type phages were recovered after the final amplification step. Aiming for a reduction of the number of cycles that could favor the selection of wild-type phages, we developed a new phage display method that leverages enzymes conjugated to the viral particles (Figure 2A,B). Usually, during PD selection, phages are exposed to the target and can be eluted after washing without knowing the exact level of phages bound to the immobilized target. The new PD protocol allowed determining the amount of phages left after each wash and enabled a decision whether to continue with the washes or proceed with the elution. In our strategy, prior to panning, M13 phages were covalently functionalized with horse radish peroxidase (HRP), which allowed monitoring in real time the level of phages bound to the surface by turnover of a fluorogenic substrate. After the washing, only the PSI-bound phages were retained in the well and we were able to quantify the amount of phages by measuring the fluorescent signal produced by the HRP. In this way, only the few phages strongly interacting with PSI were collected, enabling the generation of aptamers against PSI in a single round of selection. Afterward, the eluted phages were used to infect Escherichia coli to amplify single-phage clones. The single clones were sequenced (see Table S1, Supporting Information) and their binding affinity toward PSI was tested. We prepared three different surfaces: the first contained PSI without any preferential orientation as utilized for the panning procedure; the second was coated with a monolayer of bovine serum albumin (BSA); and the third was decorated with PSI trimers oriented with the help of an anti-PsaC antibody (which can orient PSI, but decouples it from the surface electronically) with the lumen side facing away from the surface and therefore exclusively accessible for binding (Figure 3B: top, middle, and bottom panels, respectively). For biophotovoltaic and biofuel cell applications involving PSI, it is necessary to immobilize the complex such that it injects electrons or holes directly in the electrode beneath, but not both, otherwise the charges will recombine at the electrode's surface, bypassing the external circuit. We ensured such orientation as follows. We first employed an antibody against PsaC that harbors the FB cluster—the final unit of the ETC where electron ejection takes place. When this antibody is immobilized, it forces the stromal side to orient toward the surface, preventing the binding of phages that recognize that part of PSI. We then performed binding studies to the three different surfaces (described above) in a modified phage-(enzyme-linked immunosorbent assay) (phage-ELISA) by incubating the surfaces in a solution of single clones of M13 conjugated to HRP. After washing, we added the fluorogenic substrate and determined the relative amount of bound phages via the fluorescence intensity of the surface in real time. By comparing the signal from the randomly oriented PSI to those bound to PsaC antibodies, we could differentiate between peptide sequences that bind PSI indiscriminately versus those that selectively bind the desired stromal side. The outcome of this binding assay is shown in Figure 3A. The sequences 11 (P1, RDQNHYMYSARV), 12 (P2, IQAGKTEHLAPD), and 88 (P3, LATTSHMFMAKG) are the ones that bind PSI at the desired site. They show high affinity against the surface with PSI randomly oriented (i.e., with both sides available for binding), but they display low affinity against the pristine substrate and low affinity against the lumen side of PSI (the opposite side of anti-PsaC antibody binding). Thus, they selectively bind on the stromal side (the same side as the anti-PsaC antibody), as evidenced by the low fluorescence signal when the antibodies and surface shield this side. After identifying peptide sequences that bind PSI, we characterized the ability of these peptides to orient PSI on a conducting substrate by c-AFM. Therefore, the peptide sequences were modified at the C-terminus with a phosphorylated serine residue allowing binding to an ITO surface.25 For immobilization, PSI trimers were first incubated with the modified peptides and subsequently drop cast on the ITO substrates. For comparison, the ITO surface was functionalized with a small molecule, dihydroxyacetone phosphate (DHAP), which forms SAMs capable of biasing the subsequent self-assembly of PSI toward one orientation as reported previously.22 To gain insights into the orientation of PSI, we performed c-AFM studies on the three different peptide-immobilized PSI layers and the DHAP directing linker as depicted in Figure 4. The orientation of PSI on a conducting surface can be determined by c-AFM, taking advantage of the tendency of the complexes to induce asymmetric I–V curves depending on their orientation. The origin of this rectification on Au substrates was recently elucidated and is most likely due to the permanent dipole of the PSI protein scaffold and not the cofactors of the ETC.26 When PSI is directed with the FB side or PsaC toward the substrate (i.e., the bottom electrode), a higher electrical current is measured at negative bias compared to the electrical current at positive bias giving rise to I–V data that are asymmetric about zero. This is the same orientation afforded by the PD-selected peptides. The same orientation was confirmed on ITO using DHAP as short linker molecule by comparing with data from biophotovoltaic devices incorporating PSI.22 In contrast, the opposite orientation, which is accessible with director SAMs of thiolates, gives rise to the opposite asymmetry. When PSI is lying with the axis of the ETC parallel to the surface (sideways) or if it is thermally denatured, current rectification is absent (i.e., the I–V data are symmetric). Guided by these observations, we analyzed the three different substrates with the three different peptides and an additional substrate bearing a scrambled sequence of peptide ALF (ALFHYNTHGSLH) acting as directors for the assembly of PSI and the short directing molecule DHAP. For convenience purposes, only the first three amino acids of each sequence will be used as identification. We used Pt/Ir probes for c-AFM studies in contact mode on a sample at zero voltage to record the topography of the PSI layers (Figure S2, Supporting Information). From these data we identified individual PSI trimers on the ITO surface as an approximately round, bright spot. We then set the c-AFM to I–V ramping to record the electrical characteristics of individual PSI trimers, examining at least 100 trimers for each of the three peptide linkers. The resulting I–V curves show orientation-dependent electrical behavior as described above. The expected, downward configuration of PSI shown in Figure 2C is preferred for all four peptides; however, as is shown in (Table 1), only peptide PSI exclusively in this configuration. The SAMs of PSI on the other three peptides also contained trimers in the parallel and configuration for can be found in Figure Supporting Information). After the orientation of PSI complexes one by one via c-AFM, we the electrical of SAMs of PSI using the same four peptides and the short linker DHAP using EGaIn as a top In a SAMs of oriented PSI using two different short orientation-dependent electrical using EGaIn as a top by measuring the charge transport these two oriented PSI layers at between and it was shown that the of charge transport PSI is most likely tunneling due to the of any thermally the same we compared the asymmetry of the characteristics of the peptides and SAMs of PSI with the peptides with PSI complexes by the of This the of the of PSI trimer gives into the of orientations and the of the peptides devices as to at By comparing between the peptides and the SAMs of PSI on the peptides we can any asymmetry to or We devices by incubating PSI with the modified peptides and subsequently the complexes ITO substrates. In to the ITO surface was functionalized with an of DHAP as described The EGaIn protocol is described in We PSI complexes on monolayers of different and a of in and measuring when PSI is not By biasing from V to V we were able to the asymmetry as at at to the of c-AFM (which is from Figure to the that is when the oxide contacts the biological complexes oriented by the peptides. Figure the in at V of a monolayer of only modified peptides versus the peptides with the PSI in are an of the of the versus asymmetry by the which are oriented they not be able to orient Thus, we the of of to the low of PSI complexes on and due to the of the and the affinity of the peptides for the complex and the surface is by we to with orientation, but a surface of PSI on could The in our is the of is for for for and for (Figure This be due to a binding of PSI to the peptides or affinity of the peptides to the substrate and therefore of PSI of all can be found in Figure Supporting Information). Figure the at V of devices comprising only peptide complexes directly randomly ITO and PSI oriented with the peptide EGaIn junctions comprising only peptides were that peptide is not as as PSI. The transport of ITO are to that and as it was to randomly oriented PSI on which a of By contrast, of all of the substrates PSI oriented with peptide was the most and In to on the PD we measured SAMs of PSI with the short directing linker DHAP. devices a of which is close to those of PSI oriented with but than PSI oriented with peptide (Figure After characterized the ability of to orient PSI and the of orientation in single complexes and large we oriented SAMs of PSI into We solar cells containing PSI as electrode these devices act as a to the of the orientation of PSI in devices In this way, we the of the near-perfect orientation of SAMs of PSI by peptides selected by PD and demonstrate that they their in The magnitude of in an organic solar cell is to the in function of the which can be modified with layers containing oriented When a PSI monolayer is electrons are directed to the side positive charges at the site. a of charge separation, a dipole between the semiconductor electrode and the organic is which the function of the is a different from the of on c-AFM and EGaIn which were measured in the is to the function between the bottom and the top the dipole orientation can be determined from the in We the same as an electrode to with the c-AFM and EGaIn We immobilized the PSI monolayer as described above. we immobilized PSI with which the of PSI trimers with the FB site downward to the surface orientation as as a We the active by a of the conjugated polymer and the acid in on the SAMs of PSI. the of device we or electrodes the by to and devices, (Figure and devices are by the of photogenerated In both current is generated in the organic which light than the PSI a the PSI as a of the about the orientation of the PSI be that the photogenerated current by PSI is small compared to the current generated by and that therefore the of PSI has only a on the photogenerated The on the however, shown in Table Information), the PSI in the device with the low function top In contrast, when the PSI is into the cells the high function top electrode), This behavior that the PSI monolayer the the function of that a large of the in the PSI are directed with FB toward ITO (Figure In this orientation, the photogenerated electrons of PSI are into the are between the peptides and scrambled ALF peptide compared to the small directing molecule For the cells the low function top incorporating was for all directing peptides a than V was (Table S2, Supporting Information). that all peptides a of PSI downward with FB site in close to the semiconductor and not to the organic compared to the directing of the small were for the solar cell configuration containing top electrode (Figure all of the peptides resulted in a than for DHAP and the largest of The solar cell the downward orientation of PSI by the selected peptides the orientation determined in the by c-AFM and EGaIn is retained in The orientation of proteins such as PSI on a surface is of to their behavior or to their charge separation in solar and cells. We used phage display to identify binders against the complex PSI. The selection was in such a that only one round of selection was necessary to binders and that these binders are directed to a desired side within this multiprotein the stromal side electrons are after 12 mer by phage enabled the of PSI with the of complexes oriented with the opposite toward a semiconductor surface after the peptides are modified with an surface anchoring group. of the peptides was able to all PSI molecules exclusively in one orientation. is that the orientation and its be characterized in the over large and in the light and that the orientation into Therefore, we characterized single PSI complex and SAMs and incorporated ITO into We determined the orientation of single complexes by conducting junctions with and in In all three the of the to the complexes or the chain as a function of the of will be directed toward these peptides for PSI in other biosolar and biofuel cells and to PSI in a which only the of functionalized peptides and their binding to PSI without any chemical this that the phage display technology can be to immobilize and orient protein in and and T. elongatus PSI and and and performed modified performed c-AFM and performed EGaIn and performed of oriented PSI monolayers in solid-state solar cells. and the and This was by the and The for and the and the for also the for is also a to our and this by the materials are and be for but are not or from than be to the The is not for the or functionality of any by the than be directed to the corresponding for the