Decorating a gold surface with molecular-level control over the positioning of DNA probes was demonstrated using a self-assembled monolayer (SAM) of wireframe DNA nanocube structures. The DNA nanocubes were specifically adsorbed and oriented using thiol-modified DNA on one face of the cube. The DNA nanocube SAM had a uniform coverage over the gold single crystal bead electrode with a separation of 20-30 nm measured by AFM. The face of the nanocube furthest from the gold surface was designed to hybridize with two different sequences of a 50 base single-stranded DNA probe that was modified with a fluorophore. The first 20 bases were hybridized with the DNA nanocube. One of a pair of FRET fluorophores was used for each probe strand. The dimensions of the nanocube controlled the relative spacing between these fluorophores. When the DNA probes were single-stranded, a FRET signal was observed. FRET decreased to background levels when a complementary DNA target was hybridized to either probe, resulting in a turn-off sensor with little cross-talk between the individual hybridization events. Hybridization isotherms for one target gave K-A = 170 pM and a detection limit <50 pM. In addition, the DNA nanocube SAM was configured to be used as a turn-on NeutrAvidin sensor using biotinylated DNA targets hybridized to each probe resulting in an increase in FRET. We show that the wireframe DNA nanocube can be an effective scaffold for preparing biosensors with controlled separation between surface-bound probes facilitating precise sensor surface design and enabling a wide range of sensing modalities with more than one signal available for correlative confirmation of the target binding.

The low power consumption of electrochromism makes it widely used in actively shaded windows and mirrors, while flexible versions are attractive for use in wearable devices. Initial demonstration of stretchable electrochromic elements promises good conformability to complex surfaces. Here, fully integrated intrinsically stretchable electrochromic devices are demonstrated as single elements and 3 × 3 displays. Conductive and electrochromic ionic liquid-doped poly(3,4-ethylenedioxythiophene) polystyrene sulfonate is combined with poly(vinyl alcohol)-based electrolyte to form complete cells. A transmission change of 15% is demonstrated, along with a reflectance change of 25% for opaque reflective devices, with <7 s switching time, even under 30% strain. Stability under both electrochemical and mechanical strain cycling is demonstrated. A passive matrix display exhibits addressability and low cross-talk under strain. Comparable optical performance to flexible electrochromics and higher deformability provide attractive qualities for use in wearable, biometric monitoring, and robotic skin devices.

The opioid overdose crisis in North America worsened during the COVID-19 pandemic, with multiple jurisdictions reporting more deaths per day due to the fentanyl-contaminated drug supply than COVID-19. The rapid quantitative detection of fentanyl in the illicit opioid drug supply or in bodily fluids at biologically relevant concentrations (i.e., <80 nM) remains a significant challenge. Electroanalytical techniques are inexpensive and can be used to rapidly detect fentanyl, but detection limits need to be improved. Herein, we detail the development of an electrochemical-based fentanyl analytical detection strategy that used a glassy carbon electrode modified with electrochemically reduced graphene oxide (ERGO) via electrophoretic deposition. The resulting surface was further electrochemically reduced in the presence of fentanyl to enhance the sensitivity. Multiple ERGO thicknesses were prepared in order to prove the versatility and ability to fine-tune the layer to the desired response. Fentanyl was detected at <10 ppb (<30 nM) with a limit of detection of 2 ppb and a calibration curve that covered 4 orders of concentration (from 1 ppb to 10 ppm). This method was sensitive to fentanyl analogues such as carfentanil. Interference from the presence of 100-fold excess of other opioids (heroin, cocaine) or substances typically found in illicit drug samples (e.g. caffeine and sucrose) was not significant.

The formation of defined surfaces consisting of photosynthetic reaction centers (RCs) in biohybrid solar cells is challenging. Here, we start with the production of engineered RCs for oriented binding. RCs are deposited onto gold electrodes, and 6-mercapto-1-hexanol (MCH) is used to displace multilayers and non-specifically adsorbed RCs. The resulting electrode surfaces are analyzed for photocurrent generation using an intensity-modulated light and lock-in amplifier. Atomic force microscopy (AFM) is used to characterize the surface and the formation of RC structural assemblies.

The photosynthetic reaction centre (RC) from Rhodobacter sphaeroides has been studied for use in biohybrid solar cells. Much of the previous work has focussed on improving photocurrent generation by loading the electrode surface with many copies of the protein resulting in multilayers. The primary disadvantage with this approach is the random orientation of proteins, with some supposedly oriented properly. We used RCs with Cys for covalent attachment to a gold electrode and for proper orientation. Areas of bare electrode surface and RCs bound non-specifically (i.e. not bound via the Cys) were competitively displaced by an insulating, non-redox layer of mercaptohexanol (MCH). The adsorbed monolayer of RCs was imaged using atomic force microscopy to detail the distribution of RCs on the gold surface for surfaces prepared with different RC deposition concentrations. Photocurrents were measured for all RC modified surfaces using a LED modulation method which enabled measurement of photocurrent in the presence of large faradaic currents from the sacrificial reactant (hydroquinone) at a variety of applied potentials.[1] The photocurrents generated from a monolayer composed of RCs and MCH resulted in consistent photocurrent currents. which enabled modeling of the photocurrent generation using the Marcus-Hush-Chidsey theory to extract a reorganization energy for this process. Multilayers of adsorbed RCs were distinctly different and revealed that the local environment in which the RCs are embedded significantly influenced photocurrent generation. [1] Jun, D.; Beatty, J. T.; Bizzotto, D. Highly Sensitive Method to Isolate Photocurrent Signals From Large Background Redox Currents on Protein‐Modified Electrodes. ChemElectroChem 2019, 6 (11), 2870–2875.

The use of biomacromolecules is a nascent development in clean alternative energies. In applications of biosensors and biophotovoltaic devices, the bacterial photosynthetic reaction center (RC) is a protein-pigment complex that has been commonly interfaced with electrodes, in large part to take advantage of the long-lived and high efficiency of charge separation. We investigated assemblies of RCs on an electrode that range from monolayer to multilayers by measuring the photocurrent produced when illuminated by an intensity-modulated excitation light source. In addition, atomic force microscopy and modeling of the photocurrent with the Marcus-Hush-Chidsey theory detailed the reorganization energy for the electron transfer process, which also revealed changes in the RC local environment due to the adsorbed conformations. The local environment in which the RCs are embedded significantly influenced photocurrent generation, which has implications for electron transfer of other biomacromolecules deposited on a surface in sensor and photovoltaic applications employing a redox electrolyte.

The efficient mechanism of light capture by photosynthetic proteins allows for energy transfer and conversion to electrochemical energy at very low light intensities. In this work, reaction center (RC) proteins, or a core complex consisting of the RC encircled by light harvesting (LH1) proteins (RC-LH1) from photosynthetic bacteria, were immobilized on an insulating layer of an ion-sensitive field-effect transistor (ISFET) to build bio-photodetectors. The orientation of the RC proteins was controlled via application of a hybrid linker made of 10-carboxydecylphosphonic acid and cytochrome c that anchored the RCs to their electron donor side. Bio-phototransistors consisting of either the core RC or the RC-LH1 core complex were tested under white and monochromic light. The difference between the dark and light currents at different wavelengths are well-matched with the absorption spectrum of the photosynthetic proteins. The results show potential for the use of photosynthetic proteins in photodetectors.

Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.

The reaction center (RC) protein from photosynthetic purple bacteria is an organic structure with the capability of absorbing photons at low light intensities and generating electron-hole pairs with a high efficiency. Application of this biomaterial for energy harvesting and sensor devices has been studied before. A key in employing RCs in an electrochemical device is to immobilize the proteins on an electrode. In this work, ion-sensitive field-effect transistors with Si3N4 and TaO2 gate insulator were tested to measure the success rate in immobilizing the proteins. The results show that by far Si3N4 is a better choice than TaO2, due to the effective self- assembly of the linker molecules. The density of the attached proteins to the Si3N4 transistor was estimated to be 5x10(9) proteins/cm(2) by analyzing the drift in the threshold voltage of the transistor. The fabricated device also presented the feasibility of using the RCs in an integrated photo-transistor.

The high quantum efficiency in converting light energy into a charge-separated state is a major advantage in using photosynthetic proteins in biophotovoltaic applications. Photocurrents are typically measured at open circuit potential (OCP), where the electrochemical redox or faradaic currents are minimized. However, at potentials far from the OCP, the photocurrents produced by the proteins may be impossible to measure against the large background current, owing to electrochemical redox reactions of charge-transfer mediators and/or sacrificial electron donors. Demonstrated here is a highly sensitive method using a sinusoidal-modulated intensity of an LED excitation light source to isolate the protein-based photocurrent component from the total current irrespective of electrode surface coverage. Using a genetically modified photochemical reaction center from Rhodobacter sphaeroides as a proof-of-concept, photocurrents up to 10(4)-10(5) orders of magnitude smaller than the background electrochemical redox current (due to redox reactions directly on the electrode surface) were measured at applied voltages >0.4 V from the OCP. The phase relationship between the optical excitation and photocurrent response was also measured and shown to be analytically useful.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies. https://doi.org/10.7554/eLife.34085.001 eLife digest Surrounding every living cell is a biological membrane that is largely impermeable to water-soluble molecules. This hydrophobic (or “water-hating”) barrier preserves the contents of the cell and also regulates how the cell interacts with its environment. This latter function is critical and relies on a class of proteins that are embedded within the membrane and are also hydrophobic. The hydrophobic nature of membrane proteins is however inconvenient for biochemical studies which usually take place in water-based solutions. Therefore, membrane proteins are under-represented in biological research compared to the water-soluble ones, even though roughly one quarter of a cell’s proteins are membrane proteins. Researchers have developed a few tricks to keep membrane proteins soluble after they have been extracted from the membrane. An old but popular technique makes use of detergents, which are chemicals with opposing hydrophobic and hydrophilic properties (hydrophilic literally means “water-loving”). However, even mild detergents can damage membrane proteins and will sometimes lead to experimental artifacts. More recent tricks to stabilize membrane proteins without detergents have been described but remain laborious, costly or difficult to perform. To overcome these limitations, Carlson et al. developed a simple method to stabilize membrane proteins without detergent. Called the “peptidisc”, the method uses multiple copies of a unique peptide – a short sequence of the building blocks of protein – that had been redesigned to have optimal hydrophobic and hydrophilic properties. The idea was that the peptides would wrap around the hydrophobic parts of the membrane protein, and shield them from the watery solution. Indeed, when Carlson et al. mixed this peptide with five different membrane proteins from bacteria, all were perfectly soluble and functional without detergent. The ideal ratio of peptide needed to form a peptidisc around each membrane protein was reached automatically, without having to test many different conditions. This indicates that the peptidisc acts like a “one size fits all” scaffold. The peptidisc is a new tool that will allow more researchers, including those who are not expert biochemists, to study membrane proteins. This will yield a better understanding of the structure of a cell’s membrane and how it interacts with the environment. Since the approach is both simple and easy to apply, more membrane proteins can now also be included in high-throughput searches for potential new drugs for various medical conditions. https://doi.org/10.7554/eLife.34085.002 Introduction Membrane proteins play essential roles, such as membrane transport, signal transduction, cell homeostasis, and energy metabolism. Despite their importance, obtaining these proteins in a stable non-aggregated state remains problematic. Membrane proteins are generally purified in detergent micelles, but these small amphipathic molecules are quite often detrimental to protein structure and activity, in addition to interfering with downstream analytical methods. This drawback has led researchers to develop detergent-free alternatives such as amphipols (Popot, 2010), SMALPs (Lee et al., 2016), saposin-lipoparticles (Frauenfeld et al., 2016), and the popular nanodisc system (Bayburt et al., 2006; Denisov et al., 2004). In the latter case, two amphipathic membrane scaffold proteins (MSPs) derived from apoA1 wrap around a small patch of lipid bilayer containing the target membrane protein (Bayburt et al., 2006; Denisov et al., 2004; Denisov and Sligar, 2016). However, in spite of an apparent simplicity, the formation of a nanodisc depends on several factors such as lipid to protein ratio, scaffold length, nature of lipids, rate of detergent removal and overall amenability of the target for re-assembly into lipid bilayer (Bayburt et al., 2006; Denisov et al., 2004; Hagn et al., 2013). The method is therefore not trivial and small deviations from optimal conditions often leads to low-efficiency reconstitution, or else liposome formation or protein aggregation (Bayburt et al., 2006). Peptides have been considered as an alternative to scaffold proteins. Peptergents (Corin et al., 2011), lipopeptides (Tao et al., 2013), nanostructured [beta]-sheet peptides (Privé, 2009), and bi-helical derivatives of the ApoA1-mimetic 18A peptide, termed ‘beltides’ (Larsen et al., 2016), have been reported in the recent years. Yet, these systems have not been widely adopted for structural or functional characterization of membrane proteins for various reasons. Peptergents and lipopeptides can solubilize membrane proteins directly from a lipid bilayer, but same as detergent these peptides form mixed micelles that readily aggregate and precipitate below a certain critical micellar concentration (Corin et al., 2011; Tao et al., 2013). Beltides were shown to trap bacteriorhodopsin in solution, but the method required prior incubation with specific amounts of lipids and the particles formed were reportedly unstable at physiological temperatures (Larsen et al., 2016). Cost and complexity of the peptide can also be problematic. Nanostructured [beta]-sheet peptides contain extended alkyl chains covalently linked to glycine residues, while lipopeptides need to be covalently linked to a lipid molecule (Tao et al., 2013; Privé, 2009). These hydrophobic peptides are also difficult to work with given their low solubility. Peptergents require titration of base to become soluble in aqueous solution (Corin et al., 2011), and [beta]-sheet peptides can form extended filament clusters without detergent (Privé, 2009). Thus, a peptide-based reconstitution method that is cost-effective, rapid, unhindered by issues of solubility and generally applicable to membrane proteins remains to be developped. We present the peptidisc. The peptidisc is made by multiple copies of an amphipathic bi-helical peptide (hereafter termed NSPr) wrapping around its target membrane protein. In contrast to the nanodisc, no additional lipids are necessary during reconstitution, except those that have co-purified with the protein (i.e. annular lipids). The peptide design we employ is a reverse version of the original nanodisc scaffold peptide (NSP), which consists of two repeats of the ApoA1-derived 18A peptide joined by a flexible linker proline (Kariyazono et al., 2016; Chung et al., 1985), in addition to two leucine residues which are substituted by phenylalanines to increase lipid affinity (Kariyazono et al., 2016; Mishra et al., 2008) (Supplementary file 1). The original NSP can stabilize lipid particles (Kariyazono et al., 2016), but issues of water-solubility and adaptability to membrane proteins were not demonstrated. We show here the NSPr design is very effective for stabilizing both α-helical and β-barrel membrane proteins of different size, topology, and complexity. Results The original NSP is able to capture lipids from a lipid bilayer and forms discoidal particles of varying size (Kariyazono et al., 2016). However, due to its hydrophobicity, the NSP peptide is difficult to solubilize; the solution is cloudy after resuspension in water (Figure 1B). To increase peptide solubility, we reversed the amino acid sequence of NSP, so that the two amphipathic helices have a slightly altered orientation, leading to a lower hydrophobic moment and higher overall electropotential (Figure 1AC, Supplementary file 1). We also forewent acetylation and amidation of the peptide termini so that modifications such biotinylation remain possible. These small modifications allowed the final design (termed NSPr) to be fully dissolved in water at concentrations up to 25 mg/ml (Figure 1B). Figure 1 Download asset Open asset Solubility test of NSP and NSPr. (A) Peptide models computed by the 3D-hydrophobic moment peptide calculator. The direction of hydrophobic moment is indicated by a red line. Peptides are oriented with their N to C-terminus from bottom (red) to top (blue). (B) Turbidity measurement of peptide suspension. The absorbance of light at 550 nm for NSP (blue squares, 15 mg/mL) and NSPr (red circles, 25 mg/mL), re-suspended in distilled water (dH2O) were compared to a dH2O control (green triangles). (C) Calculated electropotential and hydrophobic moment of peptide variants. Calculations were performed using the 3D-hydrophobic moment peptide calculator as described in Materials and methods. https://doi.org/10.7554/eLife.34085.003 We tested the ability of NSPr to capture the ABC transporter MalFGK2 using an ‘on-column’ reconstitution method (Figure 2). The NSPr peptide was mixed with MalFGK2 in dodecyl maltoside and the mixture applied immediately onto a size exclusion column equilibrated in a detergent-free buffer (Figure 2A). The collected particles (hereafter termed peptidisc or MalFGK2-NSPr) were soluble and monodisperse, as shown by clear-native CN-PAGE and blue-native BN-PAGE (Figure 2B). We determined the approximate peptide content using SDS-PAGE (Figure 3A). Also, since annular lipids are tightly bound to membrane proteins (Bechara et al., 2015), we determined the final lipid content in the peptidisc using thin layer chromatography and photocolorimetric methods (Figure 4A and B). This analysis indicated a stoichiometry of 10 ± 2 peptides and 41 ± 10 lipids per MalFGK2 (Table 1). This stoichiometry allowed us to calculate the mass of the MalFGK2 peptidisc to 251 ± 12 kDa (Table 1). Interestingly, the lipids identified in the TLC analysis were predominantly negative phospholipids, cardiolipin and phosphatidylglycerol (Figure 4B). These lipids play a role in regulation of MalFGK2 by stabilizing interactions with the regulatory protein EIIA (Bao and Duong, 2013). To corroborate the peptide and lipid stoichiometry, we determined the molecular weight of the intact complex by native mass spectrometry (247 ± 24 kDa; Table 1, Figure 5A and B), and size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) (250 ± 17 kDa; Table 2, Figure 6A). The SEC-MALS analysis showed that the peptidisc remains perfectly stable during storage (e.g. 3 days at 4°C). We then examined the particles by single particle negative-stain electron microscopy (Figure 2C). The 2D-class averages revealed a structure very similar to MalFGK2 in nanodiscs, (Fabre et al., 2017) with distinctly visible elements such as the MalK2 dimer, the periplasmic P2 loop and a larger discoidal density corresponding to the NSPr peptides wrapping around the MalFG membrane domain. The measured diameter of the peptidisc was 11.7 ± 1.4 nm, which is consistent with a stoichiometry of 12 ± 2 peptides per MalFGK2 complex when arranged in the double-belt model (Table 2). Finally, the ATPase activity of MalFGK2 in peptidisc was similar to that reported in proteoliposomes and in nanodiscs (Bao and Duong, 2012), in sharp contrast to the high and unregulated ATPase activity observed in detergent micelles (Figure 2D). Importantly, the structural integrity of the peptidisc remained stable at elevated temperatures, with ~80% of MalFGK2 peptidisc intact after incubation for 3 hr at 30°C (Figure 6B). Figure 2 Download asset Open asset The ‘on-column’ reconstitution of MalFGK2. (A) Typical size-exclusion chromatography of MalFGK2 in peptidisc (MalFGK2-NSPr) using the ‘on-column’ method. (B) CN-PAGE and BN-PAGE analysis of MalFGK2 in detergent micelle (DDM), nanodisc (MSP1D1), and peptidisc (NSPr). (C) Top panel: Field of view of particles stained with uranyl formate. Bottom panel: Selected class averages representing three characteristic views of MalFGK2 in peptidisc. The nucleotide-binding domains (MalK2), the transmembrane domain (MalFG), and periplasmic P2-loop are indicated with yellow, red and blue arrows, respectively. (D) Maltose-dependent ATPase activity of MalFGK2 (0.5 µM) reconstituted in detergent (DDM), proteoliposomes (PL), peptidiscs (NSPr), and nanodiscs (MSP1D1) obtained at 30°C in the presence or absence of MalE (2.5 µM). Error bars represent standard deviations from three separate experiments. https://doi.org/10.7554/eLife.34085.004 Figure 3 Download asset Open asset Quantification of NSPr in peptidiscs. (A) Left panel; 15% SDS-PAGE analysis of MalFGK2 in peptidisc or DDM. NSPr runs at the bottom of the gel and can be visualized with Coomassie blue staining. Dye fluorescence was measured on a LICOR Odyssey scanner and quantified by Image J. Right panel; Standard curve derived from NSPr titration measurement (black dots), and average intensity of NSPr fluorescence from MalFGK2 peptidisc (red dot). (B) Left Panel: Western Blot of FhuA-peptidisc reconstituted into NSPrbio, and visualized by incubation with Streptavidin-Alexa 680. Fluorescence of the Alexa 680 dye was measured on a LICOR Odyssey scanner (700 nm, excitation 680 nm) and quantified in Image J. Right Panel: Standard curve as in A. (C) 15% SDS-PAGE analysis of BRC in peptidisc. The MLH subunits of BRC partially resist denaturation by SDS, resulting in a higher molecular weight band located above the single subunits. Each gel was repeated in triplicate with independent standard curves to calculate the values reported in Table 1. https://doi.org/10.7554/eLife.34085.005 Figure 4 Download asset Open asset Quantification of phospholipids trapped in peptidiscs. (A) Calculated number of phospholipids per peptidisc. Phospholipid content was determined by Malachite green assay after acid digestion of lipid extracts. Error bars represent standard deviation derived from three separate measurements. B) TLC analysis of lipid extracts obtained from 10 µg MalFGK2 peptidisc, 10 µg FhuA peptidiscs and 20 µg BRC peptidiscs, as well as pure lipid standards Cardiolipin (CL), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (PG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE). https://doi.org/10.7554/eLife.34085.006 Figure 5 Download asset Open asset Native mass spectrometry of intact peptidiscs. Panels A, C, E are mass spectra acquired in positive ion mode for aqueous ammonium acetate solutions (100 mM, pH 7, 22°C) of MalFGK2-NSPr, BRC-NSPr and FhuA-NSPr, respectively. Panels B, D, and F are deconvoluted mass spectra of the peptidiscs shown in A, C, and E, respectively. https://doi.org/10.7554/eLife.34085.007 Figure 6 Download asset Open asset Stability of MalFGK2 peptidisc. (A) Multi-angle light scattering analysis of MalFGK2 reconstituted in peptidisc. MalFGK2-NSPr (100 µg) was left for 3 days at 4°C before analysis by SEC-MALS. Protein sample was injected and protein concentration tracked through differential refractive interferometry (dRI, black trace). Molecular weight was calculated for the fractions corresponding to the peak of MalFGK2-NSP (red trace). (B) Structural stability of MalFGK2-NSPr. The MalFGK2 peptidisc was incubated at 30°C in Buffer A for the indicated time, then analyzed by BN-PAGE. https://doi.org/10.7554/eLife.34085.008 Table 1 Calculated and observed molecular weight and scaffold stoichiometry of peptidiscs https://doi.org/10.7554/eLife.34085.009 PeptidiscMolecular weight measured by ESI-MS (kDa)Measured NSPr stoichiometry (NSPr/disc)Measured lipid stoichiometry (Lipid/Disc)Calculated molecular weight* (kDa)MalFGK2-NSPr247 ± 2410 (±2): 141 (±10): 1251 ± 12BRC-NSPr138 ± 189 (±1): 14 (±1): 1138 ± 5FhuA-NSPr137 ± 1810 (±2): 18 (±3): 1131 ± 9 *The formula for the calculated molecular weight is as follows: MWpeptidisc = MW(protein)+n(MWNSPr)+m(MWLipid); where n is the measured NSPr stoichiometry, m is the measured lipid stoichiometry, MWLipid = 0.8 kDa, MWNSPr = 4.5 kDa, and MWprotein = 173 kDa, 80 kDa, and 94 kDa for MalFGK2, FhuA, and BRC, respectively. For NSPr and Lipid stoichiometry, the standard deviation is derived from three separate measurements. Table 2 Molecular weight, diameter, and scaffold stoichiometry of MalFGK2 reconstituted in peptidisc. https://doi.org/10.7554/eLife.34085.010 Measured molecular weight (kDa)*Measured diameter †Calculated stoichiometry (scaffold/disc)‡MalFGK2 Peptidisc250 ± 1711.7 ± 1.412 (±2):1 *Molecular weight calculated from SEC-MALS data (Fig. S1). The standard error is derived from three independent SEC-MALS experiments. †Diameter of MalFGK2-peptidiscs determined by negative stain electron microscopy Figure 1B, assuming a perfectly circular shape. ‡Stoichiometry (n), based on the measured diameter of the particles, was calculated with the following formula: π(ddisc-2dα-helix) = (n/2)LNSPr; where dα-helix represents the diameter of an alpha-helix (0.5 nm), ddisc represents the measured disc diameter, and LNSPr represents length of the NSPr peptide. We also incorporated β-barrel membrane proteins in peptidisc, using the FhuA receptor as a model protein (Figure 7A). Analysis of FhuA-peptidisc by native mass spectrometry indicated a molecular weight of 138 ± 17 kDa (Figure 5E and F). Quantitation of the individual peptide and lipid components, following the approach applied to MalFGK2 above, indicated an average of 8 ± 3 lipids and 10 ± 2 NSPr per FhuA (Table 1, Figure 3B and Figure 4A). These measurements were used to estimate a molecular mass of 131 ± 9 kDa (Table 1). Binding analysis on CN-PAGE further showed that FhuA in peptidisc is functional for both TonB and colicin M (Figure 7B). The binding of TonB and colicin M is modulated by the ligand ferricrocin (Figure 7B), as previously reported in vivo and in vitro (Mills et al., 2014; Wayne et al., 1976). The peptidisc is therefore suitable for the functional reconstitution of both α-helical and β-barrel membrane proteins. Figure 7 Download asset Open asset Binding activity of FhuA in nanodiscs and peptidiscs. (A) Typical SEC profile of FhuA reconstituted in peptidisc (FhuA-NSPr) using an ‘on-column’ reconstitution protocol as described in Materials and methods. (B) The FhuA transporter reconstituted in nanodiscs (FhuA-MSPL156) or peptidiscs (FhuA-NSPr) was incubated with the C-terminal TonB23-329 fragment (2 µg) or with colicin M (5 µg), with or without ferricrocin as indicated. Samples were analyzed by CN-PAGE and Coomassie-blue staining of the gel. https://doi.org/10.7554/eLife.34085.011 The ‘in-gel’ method (Figure 8) was developed to determine optimal reconstitution conditions in a time- and cost-effective manner. Small amounts of peptides (0–2.5 µg) were mixed with the target protein (~1.25 µg) in detergent solution, and the resulting mixture immediately loaded on native gel. In that case, removal of the non-ionic detergent occurs during electrophoresis when the protein-peptide mixture enters the detergent-free part of the gel. At the correct NSP ratio, the target membrane protein does not aggregate at the top of the gel but instead migrates in a soluble form to its expected molecular weight position. This simple method allowed us to estimate the effective NSP concentrations required to trap four different integral membrane complexes into a peptidisc: MalFGK2 (Figure 8A), FhuA (Figure 8B), the trimeric OmpF porin (Figure 8C), and the membrane translocon SecYEG (Figure 8D). These membrane proteins were generally reconstituted at similar peptide concentrations, with a half-maximal molar ratio (RR50) of 20 (Figure 8E and F). This is significantly higher than the measured stoichiometry of ~10 peptides per protein complex (Table 1), suggesting that excess peptide is needed to achieve efficient assembly. This analysis also showed that the SecYEG complex can be trapped as a dimer and higher order oligomeric form in peptidisc (Figure 8D), probably due to the self-association of this complex in detergent solution, as shown before (Bessonneau et al., 2002). This later observation further differentiates the peptidisc from the nanodisc. In the nanodisc, the selective reconstitution of the SecYEG monomer and dimer requires MSP proteins of different lengths (Figure 8—figure supplement 1) (Dalal et al., 2012). Figure 8 with 1 supplement see all Download asset Open asset Express ‘in-gel’ method for determining optimal reconstitution ratio. (A) NSP and MalFGK2 were mixed at the indicated molar ratio for 2 min in Buffer A containing a low amount of detergent (~0.008% DDM) before loading onto CN-PAGE. Peptidisc reconstitution occurs during migration in the detergent-free gel environment. The same experiment was performed with. (B) FhuA,. (C) OmpF3,. (D) SecYEG. (E) Reconstitution efficiency of FhuA as a function of the NSP concentration. The protein band FhuA-NSP in B) was quantified with Image J and the data plotted as log (mol NSP/mol FhuA). The data were fitted with a Boltzmann sigmoidal function to generate a curve describing the reconstitution efficiency and the half-maximal reconstitution ratio (RR50). (F) The RR50 was determined for other target proteins as in (E). Error bars represent the standard deviation from three separate reconstitution experiments. https://doi.org/10.7554/eLife.34085.012 To save on peptide consumption, as well as to minimize exposure of the target to detergent, we developed the ‘on-beads’ reconstitution method (Figure 9), wherein affinity-purification of the protein and incorporation into peptidisc are carried out simultaneously. As illustrated in Figure 9A, the peptide was added in excess while the membrane protein still bound to the beads, followed by detergent dilution and eventually elution in a detergent-free buffer (Figure 9A, step 5). The method was tested using the his-tagged MalFGK2 complex (Figure 9B). Analysis of the eluted complex by BN-PAGE and CN-PAGE showed that MalFGK2 is readily incorporated into peptidiscs (Figure 9C), with purity and yield as good as with conventional detergent-based chromatography (Figure 9C). As an added advantage, the excess peptide collected before the elution step was re-used for the next round of ‘on-beads’ reconstitution, thereby reducing the cost of large-scale reconstitution. Figure 9 Download asset Open asset Direct ‘on-beads’ reconstitution during membrane protein purification. (A) Principle of the ‘on-beads’ reconstitution. Step 1: the tagged protein is extracted from the membrane with excess of detergent buffer (>CMC) and incubated with the affinity resin. Step 2: The beads are washed twice with the detergent buffer near its critical micelle concentration (~CMC). Step 3: The beads are incubated with buffer containing excess NSP and limited amount of detergent (

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment–protein complex. Direct electron transfer is shown to occur from the QA quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of QB quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a ~ 25 Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-β-d-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of \({{\text{P}}^{\text{+}}}{{\text{Q}}_{\text{A}}^-}\). The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the QA quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~ 12 Å. Steady-state photocurrents of up to around 200 nA/cm2 were generated in the presence of 20 mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.

The photosynthetic complexes of the thermophile Thermochromatium tepidum are of considerable interest in biohybrid solar cell applications because of the ability of thermophilic proteins to tolerate elevated temperatures. Synthetic operons encoding reaction center (RC) and light harvesting 1 (LH1) pigment-protein complexes of T. tepidum were expressed in the mesophile Rhodobacter sphaeroides The T. tepidum RC (TRC) was assembled and was found to be functional with the addition of menadione to populate the QA pocket. The production of T. tepidum LH1 (TLH1) was increased by selection of a phototrophy-capable mutant after UV irradiation mutagenesis, which yielded a hybrid RC-TLH1 core complex consisting of the R. sphaeroides RC and T. tepidum TLH1, confirmed by the absorbance peak of TLH1 at 915 nm. Affinity chromatography partial purification and subsequent sucrose gradient analysis of the hybrid RC-TLH1 core complex indicated that this core complex assembled as a monomer. Furthermore, the RC-TLH1 hybrid core complex was more tolerant of a temperature of 70°C than the R. sphaeroides RC-LH1 core complexes in both the dimeric and monomeric forms; after 1 h, the hybrid complex retained 58% of the initial starting value, compared to values of 11% and 53% for the R. sphaeroides RC-LH1 dimer and monomer forms, respectively.IMPORTANCE This work is important because it is a new approach to bioengineering of photosynthesis proteins for potential use in biophotovoltaic solar energy capture. The work establishes a proof of principle for future biohybrid solar cell applications.

Due to the high internal quantum efficiency, reaction center (RC) proteins from photosynthetic organisms have been studied in various bio-photoelectrochemical devices for solar energy harvesting. In vivo, RC and cytochrome c (cyt c; a component of the biological electron transport chain) can form a cocomplex via interprotein docking. This mechanism can be used in vitro for efficient electron transfer from an electrode to the RC in a bio-photoelectrochemical device. Hence, the success rate in coupling RCs to cyt c is of great importance for practical applications in the future. In this work, we use an electrochemical transistor to study the binding of the RC to cytochrome. The shift in the transistor threshold voltage was measured in the dark and under illumination to estimate the density of cytochrome and coupled RCs on the gate of the transistor. The results show that ~33% of the cyt cs on the transistor gate were able to effectively couple with RCs. Due to the high sensitivity of the transistor, the approach can be used to make photosensors for detecting low light intensities.

Harvesting solar energy as a carbon free source can be a promising solution to the energy crisis and environmental pollution. Biophotovoltaics seek to mimic photosynthesis to harvest solar energy and to take advantage of the low material costs, negative carbon footprint, and material abundance. In the current study, we report on a combination of zinc oxide (ZnO) nanowires with monolayers of photosynthetic reaction centers which are self-assembled, via a cytochrome c linker, as photoactive electrode. In a three-probe biophotovoltaics cell, a photocurrent density of 5.5 μA cm-2 and photovoltage of 36 mV was achieved, using methyl viologen as a redox mediator in the electrolyte. Using ferrocene as a redox mediator a transient photocurrent density of 8.0 μA cm-2 was obtained, which stabilized at 6.4 μA cm-2 after 20 s. In-depth electronic structure characterization using photoemission spectroscopy in conjunction with electrochemical analysis suggests that the fabricated photoactive electrode can provide a proper electronic path for electron transport all the way from the conduction band of the ZnO nanowires, through the protein linker to the RC, and ultimately via redox mediator to the counter electrode.

Bacterial photosynthetic reaction centers (RCs) are promising materials for solar energy harvesting, due to their high ratio of photogenerated electrons to absorbed photons and long recombination time of generated charges. In this work, photoactive electrodes were prepared from a bacterial RC-light-harvesting 1 (LH1) core complex, where the RC is encircled by the LH1 antenna, to increase light capture. A simple immobilization method was used to prepare RC-LH1 photoactive layer. Herein, we demonstrate that the combination of pretreatment of the RC-LH1 protein complexes with quinone and the immobilization method results in biophotoelectrochemical cells with a large peak transient photocurrent density and photocurrent response of 7.1 and 3.5 μA cm–2, respectively. The current study with monochromatic excitation showed maximum external quantum efficiency (EQE) and photocurrent density of 0.21% and 2 μA cm–2, respectively, with illumination power of ∼6 mW cm–2 at ∼875 nm, under ambient conditions. This work provides new directions to higher performance biophotoelectrochemical cells as well as possibly other applications of this broadly functional photoactive material.

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.

Engineered cysteine residues near the primary electron donor (P) of the reaction center from the purple photosynthetic bacterium Rhodobacter sphaeroides were covalently conjugated to each of several dye molecules in order to explore the geometric design and spectral requirements for energy transfer between an artificial antenna system and the reaction center. An average of 2.5 fluorescent dye molecules were attached at specific locations near P. The enhanced absorbance cross-section afforded by conjugation of Alexa Fluor 660 dyes resulted in a 2.2-fold increase in the formation of reaction center charge-separated state upon intensity-limited excitation at 650 nm. The effective increase in absorbance cross-section resulting from the conjugation of two other dyes, Alexa Fluor 647 and Alexa Fluor 750, was also investigated. The key parameters that dictate the efficiency of dye-to-reaction center energy transfer and subsequent charge separation were examined using both steady-state and time-resolved fluorescence spectroscopy as well as transient absorbance spectroscopy techniques. An understanding of these parameters is an important first step toward developing more complex model light-harvesting systems integrated with reaction centers.

The growing demand for nonfossil fuel-based energy production has drawn attention to the utilization of natural proteins such as photosynthetic reaction center (RC) protein complexes to harvest solar energy. The current study reports on an immobilization method to bind the wild type Rhodobacter sphaeroides RC from the primary donor side onto a Au electrode using an immobilized cytochrome c (cyt c) protein via a docking mechanism. The new structure has been assembled on a Au electrode by layer-by-layer deposition of a carboxylic acid-terminated alkanethiol (HOOC (CH2)(5)S) self-assembled monolayer (SAM), and layers of cyt c and RC. The Au|SAM|cyt c|RC working electrode was applied in a three-probe electrochemical cell where a peak cathodic photocurrent density of 0.5 mu A cm(2) was achieved. Further electrochemical study of the Au|SAM|cyt c|RC structure demonstrated similar to 70% RC surface coverage. To understand the limitations in the electron transfer through the linker structure, a detailed energy study of the SAM and cyt c was performed using photochronoamperometry, ellipsometry, photoemission spectroscopy, and cyclic voltammetry (CV). Using a simple rectangle energy barrier model, it was found that the electrode work function and the large barrier of the SAM are accountable for the low conductance in the devised linker structure.