Sortase-Mediated Ligation of PsaE-Modified Photosystem I from

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Sortase-mediated Ligation of PsaE-modified Photosystem I from Synechocystis sp. PCC 6803 to a Conductive Surface for Enhanced Photocurrent Production on a Gold Electrode Paul Frymier Langmuir, Just Accepted Manuscript • DOI: 10.1021/la5031284 • Publication Date (Web): 23 Dec 2014 Downloaded from http://pubs.acs.org on December 27, 2014

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Sortase-mediated Ligation of PsaE-modified Photosystem I from Synechocystis sp. PCC 6803 to a Conductive Surface for Enhanced Photocurrent Production on a Gold Electrode

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Langmuir la-2014-031284.R1 Article 16-Dec-2014 Le, Rosemary; University of Tennessee, Chemical and Biomolecular Engineering Raeeszadeh-Sarmazdeh, Maryam; University of Tennessee at Knoxville, CBE Boder, Eric; University of Tennessee, Department of Chemical & Biomolecular Eng. Frymier, Paul; University of Tennessee, Knoxville, Department of Chemical Engineering

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Sortase-mediated Ligation of PsaE-modified Photosystem I from Synechocystis sp. PCC 6803 to a Conductive Surface for Enhanced Photocurrent Production on a Gold Electrode Rosemary K. Le,1 Maryam Raeeszadeh-Sarmazdeh,1,2 Eric T. Boder,1,2 and Paul D. Frymier1,3* 1

Department of Chemical and Biomolecular Engineering, 2Institute for Biomedical Engineering, 3

Bredesen Center for Interdisciplinary Research and Graduate Education University of Tennessee – Knoxville, Knoxville, TN 37966-2200


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Abstract

Sortase-mediated ligation was used to attach the photosystem I (PSI) complex from Synechocystis sp. PCC 6803 in a preferential orientation to enhance photo-induced electron flow to a conductive gold surface. Ideally, this method can result in a uniform monolayer of protein, covalently bound unidirectionally to the electrode surface. The exposed C-termini of the psaE subunits of the PSI trimer were targeted to contain a LPETG-sortase recognition sequence to increase non-competing electron transfer by uniformly orienting PSI stromal side proximal to the surface. Surface characterization with atomic force microscopy suggested monolayer formation and optimal surface coverage occurred when the gold surfaces were incubated with peptide at 100 to 500 µM concentrations. Using photochronoamperometry with potassium ferro- and ferricyanide as redox mediators photocurrents in the range of 100 to 200 nA/cm2 were produced, which is an improvement over other attachment techniques for photosystem monolayers that produce approximately 100 nA/cm2 or less. This work demonstrated that sortase-mediated ligation aided in the control of PSI orientation on modified gold surfaces with a distribution of 94% stromal side proximal and 6% lumenal side proximal to the surface for current producing PSI.


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Introduction In recent years, exploration of bio-based photovoltaic technology utilizing photosynthetic reaction centers has become popular in the renewable energy field and may prove to be a viable, inexpensive alternative to traditional photovoltaics. Though bio-photovoltaics need to prove themselves against current photovoltaic technology, which have efficiencies at least two orders of magnitude greater than the best photosynthetic-based materials,1 the abundance of these photosynthetic reaction materials have their advantages. With a quantum efficiency of near unity2 and 1 V potential, the photosynthetic reaction center, photosystem I (PSI), is an ideal candidate for incorporation into a steady-state photovoltaic device. Inexpensively and easily extracted from natural cellular hosts, PSI can be adsorbed or linked to conductive or semiconductive materials to produce electricity.3-10 PSI is a large pigment protein complex involved in oxygenic photosynthesis in cyanobacteria, algae, and plants. It utilizes light absorbing cofactors like chlorophyll and carotenoid molecules, which are excited by light, to transfer electrons and convert excitation energy into chemical energy. PSI contains a reaction center (P700/P700*) that is a powerful biological reductant. With a midpoint potential of -1.3 V11 in its charge-separated state, there is considerable interest in using PSI for photocurrent generation. Integrating redox proteins like PSI into photoelectrochemical cells requires a strategy that allows for enhanced direct or mediated electron transfer between the active sites of PSI to an electrode surface by minimizing the transfer distance since most electron transfer loss is due to charge recombination.12 Furthermore, for efficient energy conversion of PSI-integrated technologies, it is important to develop technologies that are reproducible and are able to control the orientation of PSI molecules on conductive substrates, while maintaining the functionality of the protein.13


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In such a process, the primary electron donor, the P700 reaction center in PSI, is excited to P700* (Em = -1.3 V vs. SHE). The excited electron in P700* is passed to the primary electron acceptors, FA/FB (Em = -520 mV/-580 mV vs. SHE), which in turn reduces ferredoxin (Em = -440 mV vs. SHE). The resulting oxidized reaction center, P700+ (Em ≈ 490 mV vs. SHE) is then reduced back to P700 (Em = 430 mV vs. SHE) after absorbing one electron from a reducing reagent, such as plastocyanin or cytochrome c.14-16

Figure 1. Flow of electron through PSI oriented lumenal side down (left) and stromal side down (right) expected for a monolayer in orientations that can produce current in the presence of an electron acceptor or donor. The robust nature coupled with the high photon-electron conversion efficiency of PSI has led to many studies integrating PSI onto solid substrates for alternative energy17 and sensing18-20 applications. Recent efforts have shown that PSI can be attached to conductive materials via selfassembled monolayers (SAMs),3-9 covalently linked to gold by targeting cysteine residues of the protein complex,21 or deposited via vacuum22 or electric field23 assist. Though these methods are


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effective for depositing uniform monolayers and multilayers of PSI, they lead to a mono- or multi-layer of PSI molecules with mixed orientation. The resulting distribution of orientation of a PSI monolayer using these methods has been estimated to be 70% of the reaction center with its FB site accessible to the conductive surface, stromal side down, and 30% with the P700 site of the reaction center accessible to the conductive surface, or lumenal side down (Figure 1), based on simple statistical arguments and assuming equivalent reaction potential of all surface lysine residues available for covalent bonding to a gold surface modified with appropriate functional groups.7 In the case of a monolayer, current production will be observed when the terminal iron sulfur cluster (FB) donates electrons to an electrode or the electrode donates electrons to P 700 and through PSI to a terminal electron acceptor. In this work, a “stromal side down” configuration was selected as the preferred orientation. However, some of the current produced in the stromal side down orientation may be canceled out by PSI molecules that are oriented such that the P700 reaction center accepts electrons from the electrode (that is, in a “lumenal side down” orientation). It has also been determined that for significant improvements in net current production, at least 90% of the complexes must be oriented in the same direction.7 When 20 – 80% of the PSI complexes are uniformly oriented, the competing directionalities of electron transfer result in relatively small net photocurrents, whether an anodic (FB proximal to the electrode) or cathodic (P700 proximal to the electrode) current is desired. Therefore, methods that favor uniform orientation of the protein complex will lead to increased current density when compared to undirected deposition techniques. To overcome the limited current densities in monolayers due to lack of protein orientation, sortase-mediated ligation (SML) was used to attach the PSI complex in a desired orientation to enhance electron transfer to a conductive gold surface to convert light energy into electrical


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energy. Sortase A (srt A) from Staphylococcus aureus (S. aureus) was used to selectively catalyze the coupling of recombinant PSI to a tri-glycine peptide decorated gold surface. The sortase family of enzymes is widespread in Gram-positive bacteria, where they are used to attach a variety of proteins to the cell wall. Sortase A is a cysteine transpeptidase, that hydrolyzes the threonine-glycine peptide linkage in a surface protein "sorting tag" (with the amino acid sequence LPXTG, where “X” is any amino acid, typically glutamic acid, E) to form a thioacyl intermediate. The intermediate undergoes a nucleophilic attack by an amino group of a (Gly)n unit (“n” signifies the number of repeats of the glycine residue, preferably n ≥ 3) creating a new peptide bond and a resulting LPXT(G)n linkage (schematic of reaction shown in Figure 2).24-30 Sortase-mediated protein immobilization on the surface was previously used to site-specifically attach proteins to the gold surface.31 In this work, the psaE subunit of PSI was modified to contain the sortase recognition sequence, LPETG, on the exposed C-termini of the trimer (PSILPETG) shown in possible orientations in Figure 3a-d. Figure 3a shows the most desirable orientation for this study, stromal side down, with the iron sulfur clusters proximal to the surface, which will donate electrons to the surface. Figure 3b shows the trimer with its lumenal surface proximal to the surface, which can produce an undesired competing current by accept electrons from the surface. Figure 3c,d shows potential edge orientations of the PSI trimer on the surface. Previous work utilizing adsorbed PSI7,

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did not consider side-oriented PSI trimers in current

production and therefore it was assumed that these orientations do not contribute to the current production in either the preferential or undesired direction and were not considered in this study. A cysteine-terminated tri-glycine peptide was used to create a tri-glycine decorated gold surface via thiol bonds between the cysteine residue and gold to be the coupling complement. PSILPETG was site-specifically ligated to gold surfaces using sortase-mediated ligation. Surfaces


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were characterized using atomic force microscopy and photochronoamperometry to correlate current density to protein orientation controlled by the sortase-mediated ligation reaction.

Figure 2. Schematic of the sortase A transpeptidation reaction with a C-terminal LPETGmodified photosystem I to ligate the protein complex to a (glycine)n decorated gold surface. Linkage shown not drawn to scale.


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Figure 3. Crystal structure of the PSI trimer of Thermosynechoccocus elongatus (PDB ID: 1JB0) as a surrogate for Synechocystis PCC 6803 PSI with the PsaE subunits highlighted in blue. The red spheres indicate the C-termini of the PsaE subunits targeted for introduction of the LPETG recognition sequence. (a) Stromal side down orientation, b) lumenal side down orientation, c) single ligation binding edge orientation, and (d) double ligation binding edge orientation.

Experimental Section Construction, Culture, and Purification of psaE-LPETG-His tagged Photosystem I PSI from the model cyanobacterium Synechocystis sp. PCC 6803 (ATCC: 27184) was engineered to express an LPETG recognition sequence necessary for sortase coupling on the exposed C-terminus of the psaE subunit. The psaE sequence from Synechocystis genomic DNA was modified by PCR to include a sequence encoding a C-terminal sortase recognition tag (LPETG) and a 6xHis-tag to facilitate protein purification. The psaE-LPETG-6xHis mutant photosystem I strain of Synechocystis PCC 6803 (6803) was generated by transforming wild-type 6803 cells with psaE-LPETG-6xHis plasmid following the protocol for construction of mutants by Eaton-Rye33 PsaE was targeted in particular due to the exposed nature of its C-terminus which should increase the likelihood that sortase would be able to access the recognition tag. Genomic DNA was extracted from wild type 6803 following the


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protocol by Eaton-Rye. The gene sequence of the psaE subunit was obtained from the online resource CyanoBase (http://genome.microbedb.jp/cyanobase), as well as the 1 kb upstream and 1 kb downstream regions of the target gene. Primers (Integrated DNA Technologies, Inc.) were designed to target the gene of interest and the respective 1 kb upstream and 1 kb downstream regions; Zang et al. have shown that with increasing homology, the transformation efficiency increases.34 The upstream forward primer (UF1) introduced an ApaI restriction site 1 kb upstream from the gene and the upstream reverse primer (UR1) introduced an AatII restriction site, a 6xHis moiety, and the LPETG sortase recognition sequence to the C-terminus of psaE (sequence shown in Table 1). The downstream primers (DF1, forward and DR1, reverse) introduced SacI and NsiI restriction sites at the N- and C-terminus of a 1 kb fragment downstream from the psaE gene, respectively. A kanamycin resistance cassette was introduced into the pGEM Easy-T vector via TA cloning kit (Promega). The target gene sequences were amplified from wild type genomic 6803 DNA using conventional PCR. Each DNA fragment was inserted into the pGEM:kanr plasmid vector and transformed into JM109 competent E. coli cells (Promega) sequentially. Plasmid DNA extracted from positive clones were sequenced to verify the correct insertion of the LPETG sequence. A 5 mL culture of transformed E. coli cells was grown, such that at least 5 µg of plasmid DNA could be recovered for transformation into wild type Synechocystis cells. A 5 mL culture of wild type Synechocystis was grown for two to three days at 30 °C, shaking at 225 rpm, under 15 µE/m2/s fluorescent light illumination until OD730 = 0.2 to 0.8. Cells were pelleted by centrifugation at 3,000 x g, 25 °C and resuspended in 0.5 mL non-selective bluegreen media (BG-11) optimized for freshwater algae and protozoa so that OD730 ~ 2.5. The cells were incubated with 5 µg of the modified psaE plasmid at 30 °C, shaking at 120 rpm, and 15


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µE/m2/s fluorescent light for 5 h. Transformed cells were pipetted onto a sterile piece of Whatman filter paper and placed on top of non-selective BG-11 agar for three days to help the cells recover before being transferred to 12.5 µg/mL kanamycin selective BG-11 agar for three days. After recovering on a half dose of selective media, the filter was transferred to full antibiotics at a concentration of 25 µg/mL kanamycin. Transformants were grown at 30 °C under 15 µE/m2/s fluorescent light illumination for all antibiotic conditions. Growth on full antibiotics took two to three weeks for colonies to appear. Colony PCR was performed on colonies that appeared on 25 µg/mL kanamycin using UF1 and DR1 primers to amplify the target subunit for sequencing. This fragment was then sequenced to verify that the sequence encoding LPETG had incorporated into the genome of Synechocystis. Further selection was performed on 50 µg/mL kanamycin to ensure complete segregation of the wild type from the mutant. Confirmed mutants were grown in 50 µg/mL kanamycin selective BG-11 media in a New Brunswick BioFlow3000 Bioreactor (Eppendorf, Inc.) at 30 °C under 25 µE/m2/s illumination with fluorescent lights, 350 rpm impeller agitation, and 150 mL/min air flow rate in a four liter volume. Cells were harvested, pelleted, and frozen at –80 °C before PSI extraction. PSI was extracted from the mutant psaE 6803 strain following modified protocols.35-38 Frozen cells (10 g) were resuspended in Buffer A (50 mM MES-NaOH, pH 6.0; 10 mM MgCl2; 5 mM CaCl2; 25% v/v glycerol) by vortexing, such that the chlorophyll a concentration was at 1 mg/mL. The concentration of chlorophyll was determined using methanol extraction, by mixing resuspended cells with 90% methanol at a 1:10 dilution. The cell-methanol mix was incubated at 60°C for two minutes and centrifuged at 21,100 x g in a microcentrifuge for 2 min. The supernatant was transferred to a 1 mL quartz cuvette, and the absorbance was measured at 665 nm with a UV-Vis spectrophotometer. The concentration of chlorophyll present in was determined by Equation 1,


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(1) where the extinction coefficient of chl a at 665 nm in methanol is 72 mg/L.39 100X HALT protease inhibitor cocktail for His-tagged proteins (Thermo Scientific) was added diluted to 1X concentration (100 µL per 10 mL cell suspension) on ice before cell lysis with a French press at 25,000 psi, for three cycles. The cell lysate was collected and spun down at 19,000 x g for 30 min at 4 °C. The supernatant was removed and the cell lysate pellet was resuspended in Buffer A and solubilized in n-dodecyl-ß-D-maltoside (DDM) at a final concentration of 1% (wt/v) for 30 min at 4 °C on a platform rocker. The solubilized solution was centrifuged at 15,000 x g for 30 min. The recovered supernatant containing the solubilized fraction of the LPETG-His-tagged PSI was run on a HisPur Cobalt (Thermo Scientific) column to separate the tagged protein from undesired proteins, 5 mL resin bed per 20 mL supernatant. The column and sample were equilibrated with Buffer A, pH 7.8, 0.04% (wt/v) DDM, 5 mM histidine. After applying the PSI sample and re-applying the flow through, the column was washed with nine bed volumes until the 280 nm UV-VIS measurement of the wash reached baseline. The protein was eluted from the column using Buffer A, pH 7.8, 0.04% (wt/v) DDM, 100 mM histidine. Elution fractions were pooled and dialyzed with 6-8000 MWCO Spectra/Por dialysis membrane tubing (Spectrum Laboratories, Inc.) in Buffer A with 0.04% (wt/v) DDM for 2 h, twice, followed by overnight dialysis at 4 °C with gentle stirring.

Sortase Expression and Purification S. aureus Sortase A was cloned out of the pHTT27 plasmid. The sortase construct was inserted into a pET-26b(+) vector using NdeI and XhoI restriction sites. A 6xHis-tag sequence was


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inserted at the N-terminus of the construct and a stop codon was placed before the XhoI restriction site to prevent expression of the second His-tag at the C-terminus. Sortase was expressed in BL21 (DE3) E. coli cells (Life Technologies). An overnight cell culture was diluted (1:50 to 1:100) in 250 mL of LB kanamycin (50 µg/mL) media. Cells were grown to an OD600 of 0.5 to 0.7 at 37 °C with shaking at 225 rpm, induced by adding IPTG (Fisher BioReagents) to a final concentration of 1 mM, and pelleted at 6000 x g for 20 min after 3-4 h of induction. The cells were frozen at –20 °C overnight. Pellets were thawed on ice and the cytosolic proteins were extracted using B-PER reagent (Thermo Scientific) with 10 μL (2 U/μL) DNase I (NEB), following manufacturer’s instructions. The cell lysate was incubated at room temperature for 10 min on ice and the soluble proteins were separated from the insoluble proteins by centrifugation at 15,000 x g for 5 min at 4 °C. The sortase-containing supernatant was recovered and sortase was purified using TALON resin (Clontech), following the manufacturer’s batch/gravity protocol. The purified protein was then dialyzed using a Slide-A-Lyzer cassette (Thermo Scientific) with 10,000 kDa MW cutoff (Thermo Scientific) or buffer exchanged against TBS buffer (50 mM Tris base, 150 mM NaCl, pH 7.5) using Amicon centrifugal filters (Millipore) with 10,000 kDa MW cutoff to remove imidazole. The protein concentration was determined using an A280 measurement with a Bio-Rad SmartSpec 3000 spectrophotometer and an extinction coefficient of 19000 M-1 cm-1. 2.5 to 10 mg of the protein was produced from 250 mL cultures. Sortase was concentrated to 500 to 1200 μM with Amicon centrifugal filters. The concentrated protein was stored in aliquots for up to six months at –20 °C for future use and at 4 °C for short-term storage.


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Preparation of Peptide and PSI Decorated Gold Surfaces Aluminosilicate glass slides coated with 10 nm gold over a 2 nm titanium adhesion layer (Platypus Technologies AU.0100.ALSI) were cut into 1.27 cm x 1.27 cm squares and cleaned in a 3:1 ammonium hydroxide: hydrogen peroxide base piranha solution for 10 min to remove organic contaminants. The slides were then rinsed thoroughly with 10 mL of oxygen-free ddH2O three times. Synthetic GGGC peptide was purchased at > 95% purity (Genscript). The peptide solution was prepared by dissolving dried peptide in an isopropanol-DMF solution with vortexing and diluting to the appropriate concentration under oxygen-free conditions to prevent disulfide bond formation between peptides in solution. Piranha-treated gold slides were immersed in 15 mL of GGGC (100 to 500 µM) peptide solution for 48 h under oxygen-free conditions. The peptide-coated substrates were rinsed with O2-free ddH2O to remove excess peptide and dried under nitrogen prior to analysis using atomic force microscopy and PSI deposition. The 1.61 cm2 peptide-coated gold substrates were incubated with a solution containing modified psaE-PSI-LPETG (100 µM chl a ~ 0.35 µM PSI trimer), sortase (5 µM), CaCl2 (10 mM), 0.04% (wt/v) DDM, and pH 7.6 TBS buffer applied as a 150 µL droplet. The substrates were placed in a petri dish wrapped with parafilm to prevent evaporation and incubated at 30 °C shielded from light for 6 h in a humidified environment. The PSI-LPETGGGC-gold surfaces were washed with ddH2O to remove excess PSI not bound by the sortase linkage and stored in 1X TBS buffer (pH = 7.6) with 0.04% (wt/v) DDM. The surfaces were dried with nitrogen before use such that a 3.2 cm piece of copper tape (JVCC) could be attached to a corner of the substrate/working electrode and folded adhesive side together, to make a sealed connection with the working electrode for use in the custom electrochemical cell.


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Atomic Force Microscopy Topographical scans were performed using an Asylum Research MFP-3D atomic force microscope with silicon Olympus AC240TS cantilevers with a spring constant of 2 (0.5 to 4.4) N/m and resonant frequency of 70 (50 to 90) kHz. Each 1.61 cm2 sample was scanned in four 20 µm by 20 µm sections to judge the uniformity of the deposition of peptide and PSI on the surface. Better resolution of the deposition was achieved by increasing the magnification to encompass 5 µm by 5 µm sections within each 400 µm2 section.

Electrochemical Measurements Photochronoamperometry measurements were made using a BioLogic SP-200 potentiostat (BioLogic) with a custom three-electrode cell configuration (provided by Kane Jennings and David Cliffel, Vanderbilt University). The gold substrate with PSI and suitable controls were used as the working electrode with a 0.385 cm2 effective working area. A saturated calomel electrode (+0.244 V vs. SHE) was used as the reference electrode and a platinum wire was used as the counter electrode. The electrochemical mediator solutions used for our experiments were 200 µM potassium ferro- or ferricyanide (K4Fe(CN)6 or K3Fe(CN)6), in 1X phosphate buffered saline (PBS = 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7), and 100 mM KCl. The electrode cell was illuminated with a Fiber-Lite DC regulated illuminator (Dolan-Jenner Industries) with an OSRAM 150 W quartz halogen lamp. The light was filtered through a 590-nm long-pass filter and provided 500 µE/m2/s (10.5 mW/m2) light intensity. The current density was calculated by taking the difference in the dark current and the current under illumination after ~15 s. Amperometry measurements were made after measuring the open circuit potential (OCP) of the cell in the dark until a baseline value was reached (~3 min). All


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measurements were made under the OCP measured for each sample (~ –100 mV vs. SCE), such that the dark current would yield a value of approximately 0 nA/cm2. Prior to illumination, the system was allowed to equilibrate to a baseline value (~0 nA/cm2) at the applied OCP for ~15 s in the dark. After equilibration the working area was illuminated for 20 s and returned to baseline for 20 s before repeating for three light-dark cycles total. The photo-catalyzed response was measured and recorded by the EC-Lab® program provided with the electrochemistry workstation.

Results and Discussion Site-Specific Immobilization of PSI-LPETG on Gold using Sortase Recombinant PSI with the psaE subunit modified to include the sortase tag LPETG was attached to a tri-glycine coated gold surface using sortase-mediated protein attachment. Incubating the substrates with the peptide created a dense monolayer of Gly3 via thiol bonds with the gold and this monolayer served as the complimentary acceptor of the sortase-catalyzed ligation of modified psaE-PSI-LPETG to the surface. AFM was used to determine approximate surface coverage of peptide and PSI psaE-LPETG on the gold substrates (Figure 4). From the AFM images, the average height for the samples with different peptide concentrations and PSI was determined. The cleaned bare gold (Figure 4a) substrates yielded surface features with an average height of 2.1 nm with 95% lower and upper bound confidence intervals of 1.3 nm and 2.9 nm, respectively. The AFM analysis of different peptide concentrations in the range of 100 to 500 µM on gold showed that the addition of peptide yields an average height of the surface features of 2.3 nm with 95% lower and upper bound confidence intervals of 1.9 nm and 2.7 nm, respectively. However, there are some features on the bare gold (Figure 4a) and peptide coated


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gold (Figure 4b) that approach 10 nm. With the addition of LPETG-modified PSI, it was expected that the surface would yield an average feature height of approximately 14 nm, where bare gold has average height features ~2 nm, the LPETGGGC linkage contributes ~2 nm, and the height of the protein, ~10 nm, which was previous observed with small-angle neutron scattering.40 The observed average height of the SML PSI surface was 9.9 nm with 95% lower and upper bound confidence intervals of 6.77 nm and 13.0 nm, respectively (Figure 4c). As in the case with bare gold and peptide covered gold, there are some features that are around 20 nm in height. However, this is consistent with a monolayer of PSI that is formed on top of an inherently variable surface. As discussed above, there are features even on the bare gold and peptide covered gold that are as much as 10 nm above the lowest areas on the image (see Figure 4a,b).

Figure 4. AFM images of SML PSI gold surface. (a) Bare gold substrate, (b) 100 µM GGG peptide on gold, and (c) PSI-LPETG on 100 µM GGG-peptide gold ligated via SML.

Electrochemical Characterization of PSI Monolayer In vivo, redox reaction across PSI are aided by diffusible mediators; cytochrome c6 on the donor side to P700 and ferredoxin on the acceptor side from the iron sulfur cluster FB. For in vitro


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applications, where PSI has been purified from the thylakoid membrane, such as deposition onto electrodes for electricity production, continuous electron transfer is maintained by either an electron acceptor or donor, depending on the orientation of PSI. In this work, potassium ferrocyanide (K4Fe(CN)6, Em ~436 mV vs. SHE) was used as an electron donor to PSI oriented with its P700 reaction site adjacent to the bulk electron mediator solution to reduce P700+ back to P700, referred to as the stromal side down orientation. The terminal electron acceptor was a gold electrode which oxidized FB. K4Fe(CN)6 was selected as an electron donor for this work because its electron donation is light-independent and its potential is compatible with donation to oxidized P700+. Determination of the orientation of PSI on the surface was done by performing photochronoamperometry measurements with PSI-gold samples in either 200 µM potassium ferri- (K3) or ferro- (K4) cyanide, with 100 mM KCl and 1X PBS (pH = 7). PSI behaves like a diode since electron flow in PSI is unidirectional. Therefore the direction of the electron flow/current can be probed by determining which species of the soluble mediator (potassium ferri- or ferro-cyanide) enables current flow. A cathodic current was designated as flow of electrons from the gold electrode through PSI to an electron accepting species in solution while an anodic current was designated as flow of electrons into the gold electrode from an electron donating species in solution through PSI. Under this designation, cathodic current was assumed to be the result of PSI with the P700 reaction site proximal (lumenal side down, Figure 1a) to the electrode while an anodic current was the result of the PSI oriented such that the FB clusters were proximal (stromal side down, Figure 1b). It was expected that a significant net current response would occur in the presence of ferrocyanide (reduced mediator/electron donor). In contrast, in the presence of ferricyanide (oxidized mediator/electron acceptor) it was expected that little to no


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Langmuir

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current would be produced, since ferricyanide is unable to donate electrons to P700 in the assumed orientation.

Figure 5. Sample photocurrent traces generated by mutant and wild-type PSI SML controls under OCP using K4Fe(CN)6 as an electron donor after one light cycle. (a) Mutant PSI SML controls. (b) WT PSI SML controls. Traces offset from zero to allow viewing of individual traces. Figure 5 shows a sampling of the raw photochronoamperometry traces of current producing controls for one light cycle, where the three-electrode system is equilibrated in the dark for 10 to 20 s, illuminated for ~20 s, then returned to dark conditions. The average current density of three samples for each control trace is summarized in Table 2, where the averaged baseline/dark current was subtracted from the averaged peak value during illumination and is shown with the standard deviation from the mean. As expected, the controls of the bare gold surface cleaned with piranha solution, with peptide alone, sortase enzyme alone, peptide and sortase, and mutant


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Langmuir

PSI alone produced no current or current that is below the detection limit of the potentiostat. The wild type PSI alone produced very little current, with an average value of 2.5 ± 4.3 nA/cm2. Interestingly, in the case of modified LPETG and wild type PSI in the presence of sortase but no GGG peptide substrate produced current in a range expected for PSI monolayers (

Sortase-Mediated Ligation of PsaE-Modified Photosystem I from

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