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

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.

Afterwards, the membrane suspension was washed once with Buffer A and three times with Buffer containing 0,05% DDM (n-dodecyl-β-D-maltoside) in order to remove the phycobilisomes. Finally, the thylakoid membranes were solubilized by incubation in Buffer A supplemented with 0,6% DDM for 30min at 20°C in the dark. Non-solubilized material was pelleted by centrifugation [JLA 16.250 rotor, Beckman;16000rpm (38000xg); 20min] and the supernatant subjected to subsequent purification steps.

Photosystem I purification
For PSI purification, fast liquid protein chromatography was applied on solubilized thylakoid membranes. The chromatographic purification was performed on a ÄKTAexplorer [GE Healthcare] using an anion exchange column [HiTrapTM Q HP, GE Healthcare]. After column equilibration with Buffer A + 0,03% DDM, the sample was applied and subsequently eluted by a linear gradient of [0-1M] MgSO 4 . The green fluorescent fractions were collected and desalted with Buffer A + 0,03% DDM using Vivaspin 20 columns [molecular weight cutoff: 100kDa; GE Healthcare]. Finally, the purified PSI sample was adjusted to a Chl a concentration of 800 µM [with Buffer A + 0,03% DDM], snap-frozen in liquid nitrogen and stored at -80°C. Determination of Chlorophyll a and Protein Concentration: Chl a determination was performed in 100% methanol as described in Porra et al. [1] Absorption and Fluorescence Spectra: All absorption and fluorescence spectra were recorded with a SpectraMax M2 spectrophotometer (Molecular Devices, US) at 25°C.

Phage Display
Chemical coupling of horse radish peroxidase (HRP) to M13 phage library: Coupling of HRP (EZ-Link TM Plus Activated Peroxidase -Thermo scientific) was performed following the vendor's protocol using 10e11 phages (NEB #E8110S) and 1mg of peroxidase. HRP was applied in excess in relation to the phage to ensure high degree of functionalization of the phage particles. The HRP, which was activated by periodate treatment at the sugar moieties to exhibit aldehyde functions, which are expected to react with amino groups on the phage surface of the pVIII coat protein. The Schiff base formation was carried out in sodium carbonate pH 9.8 at 16°C for 16 h in a shaker at 700 rpm. Then, reductive amination was induced by addition of 10 μl of sodium cyanoborohydride (5 M) to the reaction mixture in order to stabilize the conjugation of HRP to M13 coat proteins. After terminating the reaction by addition of ethanolamine 3 M pH 10, the excess of unreacted HRP was separated from the phages by filtration using a 50.000 Da cutoff spin column. The flow-through (i.e. the HRP) was further tested for nonspecific adsorption to the functionalized plates to verify any level of background signal due to unreacted HRP in the biopanning experiment. No background noise was detected (data not shown).
Both the supernatant (containing the phages conjugated to HRP) and the flow-through (containing quenched HRP) were tested for infectivity and peroxidase activity. As expected, only the phage-containing fraction was infective and both fractions showed high HRP activity.
These results prove that the phage particles conjugated to peroxidase are still infective and HRP is still catalytically active, two criteria essential for our selection system.

Single round phage display (PD).
A single round PD selection was carried out using HRPconjugated phage particles. The original library of phages expressing 12mer peptides at the Nterminus of their pIII protein was purchased from NEB (NEB #E8110S). After HRPconjugation, a population of 10 9 phages per well was selected to be exposed to PSI-modified surfaces. The wells of the plate were prepared according to the manufacturer's instruction (Nunc™ Amino™ Immobilizer Surface, Thermo Scientific). In brief, the wells were incubated with 100 μl of a solution of PSI (300 μM) with gentle agitation (200 rpm) at RT over-night. The wells were then aspirated and washed three times with PBST containing 1% BSA (w/v).
In previous experiments, we observed some binding affinity of the phage library to the pristine plate, probably due to plastic binders being present in the library. Therefore, an initial negative selection against blank wells was performed. Next, the phages were split in two sub populations and incubated separately in wells with and without PSI target. After extensive washing steps using PBST, peroxidase substrate (QuantaBlue Fluorogenic Peroxidase Substrate Kit-ThermoFisher #15169) was added to each well to determine the presence of bound phages. When a signal from the blank wells was absent and a clear signal originating from the PSI containing wells was detected, elution with Tris-glycine buffer was initiated.
Following the same procedure, the elution step was monitored to prevent phages remaining bound to the target. Before and after elution, the developing buffer (QuantaBlu™ Fluorogenic Peroxidase Substrate Kit) for the HRP was added to the wells and the activity of peroxidase was measured over time in RFU.

Conducting AFM measurements.
The height images of PSI were measured in tapping mode with TESP-V2 probes by a MultiMode 8 AFM Microscope with System Controller V. External TUNA mode was used for the height and electrical I-V characteristics recording in contact mode. ANSCM-PC probes with a spring constant of k = 0.2 N/m, tip radius 30 nm and tip height 2.5-8 µm were utilized.
The scanning region was selected with a size of up to 2 µm by 2 µm. The scanning rate and number of lines were selected to be 0.2 Hz and 512 lines/sample, respectively. For each image and electrical characterizations, a new probe was employed to avoid AFM tip broadening due to wear and contaminations from the measuring surface. The AFM images were analyzed with NanoScopeAnalysis software.

Fig. S1. Readout of the phage display against PSI carried out with HRP conjugated M13
PhD 12. Signal of the phages bound to the negative control wells after incubation (green line). Unbound phages were transferred in wells with and without PSI and washed (violet and blue lines, respectively). The yellow and red lines represent the signal from blank well and PSI after elution. Fluorescence intensities are the average of three measurements with corresponding error bars.