Lipids and block copolymers can individually self-assemble into vesicles, each with their own particular benefits and limitations. Combining polymers with lipids allows for further optimisation of ...the vesicle membranes for bionanotechnology applications. Here, POPC lipid is mixed with poly(1,2-butadiene-
-ethylene oxide) of two different molecular weights (PBd
-PEO
, Mr = 1800 g mol
and PBd
-PEO
, Mr = 1150 g mol
) in order to investigate how increasing the polymer fraction affects membrane mixing, hydration and fluidity. Intensity contributions of fluorescently labelled lipid and polymer within mixed GUV membranes confirm membrane homogeneity within the hybrids. General polarisation measurements of Laurdan in GUVs showed little change in membrane hydration as polymer fraction is increased, which suggests good structural compatibility between lipids and polymers that gives rise to well-mixed vesicles. Membrane fluidity in hybrid GUVs was found to decrease non-linearly with increasing polymer fraction. However, the diffusion coefficients for the fluorescent polymer in hybrid membranes did not change significantly with increasing polymer content. While increasing the polymer fraction does reduce the movement of lipids through a polymer-rich matrix, insignificant difference in diffusion coefficients of the polymer suggests that its diffusion is minimally affected by increasing lipid composition in the range studied. These results lay further foundations for the wider development of hybrid vesicles with controlled properties for advanced biotechnologies.
For more than three decades, the field of bioelectrochemistry has provided novel insights into the catalytic mechanisms of enzymes, the principles that govern biological electron transfer, and has ...elucidated the basic principles for bioelectrocatalytic systems. Progress in biochemistry, bionanotechnology, and our ever increasing ability to control the chemistry and structure of electrode surfaces has enabled the study of ever more complex systems with bioelectrochemistry. This feature article highlights developments over the last decade, where supramolecular approaches have been employed to develop electrode assemblies that increase enzyme loading on the electrode or create more biocompatible environments for membrane enzymes. Two approaches are particularly highlighted: the use of layer-by-layer assembly, and the modification of electrodes with planar lipid membranes.
Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as
and
, are Gram negative. ...More recently, it has been reported that Gram-positive bacteria, such as
and
, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of
, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria.
Bio-hybrid nanomaterials have great potential for combining the most desirable aspects of biomolecules and the contemporary concepts of nanotechnology to create highly efficient light-harvesting ...materials. Light-harvesting proteins are optimized to absorb and transfer solar energy with remarkable efficiency but have a spectral range that is limited by their natural pigment complement. Herein, we present the development of model membranes ("proteoliposomes") in which the absorption range of the membrane protein Light-Harvesting Complex II (LHCII) is effectively enhanced by the addition of lipid-tethered Texas Red (TR) chromophores. Energy transfer from TR to LHCII is observed with up to 94% efficiency and increased LHCII fluorescence of up to three-fold when excited in the region of lowest natural absorption. The new self-assembly procedure offers the modularity to control the concentrations incorporated of TR and LHCII, allowing energy transfer and fluorescence to be tuned. Fluorescence Lifetime Imaging Microscopy provides single-proteoliposome-level quantification of energy transfer efficiency and confirms that functionality is retained on surfaces. Designer proteoliposomes could act as a controllable light-harvesting nanomaterial and are a promising step in the development of bio-hybrid light-harvesting systems.
The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution ...compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm–2 at 0.4 V vs SHE could be obtained at pH 6.5 (E onset = 0.72 V). UV–visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors.
Protein–protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein–protein interactions regulate the activity of a respiratory-chain ...enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis. We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein–protein interactions.
CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from ...menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately -240 mV) and low-spin (approximately -110, -190 and -265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E(m) = -80 mV) in the presence of NADH (E(m) = -320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.
Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles ...of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 μg/ml, but DNA damage was evident at 0.1 μg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 μg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles.
C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient ...for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.
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