Pulsed dipolar spectroscopy, such as double electron-electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. ...In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate the effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function.
DEERefiner provides an efficient approach for modeling atomic-level protein structures, notably showcasing its effectiveness on the multidrug transporter LmrP using pulsed dipolar spectroscopy data.
This work confirms the presence of a large facet‐dependent photocatalytic activity of Cu2O crystals through sparse deposition of gold particles on Cu2O cubes, octahedra, and rhombic dodecahedra. ...Au‐decorated Cu2O rhombic dodecahedra and octahedra showed greatly enhanced photodegradation rates of methyl orange resulting from a better separation of the photogenerated electrons and holes, with the rhombic dodecahedra giving the best efficiency. Au–Cu2O core–shell rhombic dodecahedra also displayed a better photocatalytic activity than pristine rhombic dodecahedra. However, Au‐deposited Cu2O cubes, pristine cubes, and Au‐deposited small nanocubes bound by entirely {100} facets are all photocatalytically inactive. X‐ray photoelectron spectra (XPS) showed identical copper peak positions for these Au‐decorated crystals. Remarkably, electron paramagnetic resonance (EPR) measurements indicated a higher production of hydroxyl radicals for the photoirradiated Cu2O rhombic dodecahedra than for the octahedra, but no radicals were produced from photoirradiated Cu2O cubes. The Cu2O {100} face may present a high energy barrier through its large band edge bending and/or electrostatic repulsion, preventing charge carriers from reaching to this surface. The conventional photocatalysis model fails in this case. The facet‐dependent photocatalytic differences should be observable in other semiconductor systems whenever a photoinduced charge‐transfer process occurs across an interface.
Structure is the key: Whereas Au‐decorated Cu2O octahedra and rhombic dodecahedra (RD) show enhanced photocatalytic activities compared to their pristine particles, small and large Au‐decorated Cu2O cubes remain inactive. EPR measurements indicate a higher production of hydroxyl radicals for Cu2O rhombic dodecahedra than for octahedra upon photoirradiation, but no radicals are produced from photoirradiated Cu2O cubes (see figure).
Caspase-8-cleaved Bid (cBid) associates with mitochondria and promotes the activation of BAX, leading to mitochondria outer membrane permeabilization (MOMP) and apoptosis. However, current structural ...models of cBid are largely based on studies using membrane vesicles and detergent micelles. Here we employ spin-label ESR and site-directed PEGylation methods to identify conformations of cBid at real mitochondrial membranes, revealing stepwise mechanisms in the activation process. Upon the binding of cBid to mitochondria, its structure is reorganized to expose the BH3 domain while leaving the structural integrity only slightly altered. The mitochondria-bound cBid is in association with Mtch2 and it remains in the primed state until interacting with BAX. The interaction subsequently triggers the fragmentation of cBid, causes large conformational changes, and promotes BAX-mediated MOMP. Our results reveal structural differences of cBid between mitochondria and other lipid-like environments and, moreover, highlight the role of the membrane binding in modifying cBid structure and assisting the inactive-to-active transition in function.
Given the huge economic burden caused by chronic and acute diseases on human beings, it is an urgent requirement of a cost-effective diagnosis and monitoring process to treat and cure the disease in ...their preliminary stage to avoid severe complications. Wearable biosensors have been developed by using numerous materials for non-invasive, wireless, and consistent human health monitoring. Graphene, a 2D nanomaterial, has received considerable attention for the development of wearable biosensors due to its outstanding physical, chemical, and structural properties. Moreover, the extremely flexible, foldable, and biocompatible nature of graphene provide a wide scope for developing wearable biosensor devices. Therefore, graphene and its derivatives could be trending materials to fabricate wearable biosensor devices for remote human health management in the near future. Various biofluids and exhaled breath contain many relevant biomarkers which can be exploited by wearable biosensors non-invasively to identify diseases. In this article, we have discussed various methodologies and strategies for synthesizing and pattering graphene. Furthermore, general sensing mechanism of biosensors, and graphene-based biosensing devices for tear, sweat, interstitial fluid (ISF), saliva, and exhaled breath have also been explored and discussed thoroughly. Finally, current challenges and future prospective of graphene-based wearable biosensors have been evaluated with conclusion.
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Graphene is a promising 2D material for the development of wearable sensors. Various biofluids (sweat, tears, saliva and ISF) and exhaled breath contains many relevant biomarkers which facilitate in identify diseases. Biosensor is made up of biological recognition element such as enzyme, antibody, nucleic acid, hormone, organelle, or complete cell and physical (transducer, amplifier), provide fast response without causing organ harm.
Bisphenol A (BPA) is an estrogen‐like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA ...residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE‐19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE‐19 cells. After BPA treatment, the expression of Bcl‐XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase‐9 to promote downstream caspase‐3 leading to cytotoxicity. The nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and heme oxygenase 1 (HO‐1) pathway play a major role in age‐related macular degeneration. Our results showed that expression of HO‐1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP‐activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA‐induced ROS generation and cytotoxicity were reduced by N‐acetyl‐l‐cysteine. Taken together, these results suggest that BPA induced ARPE‐19 cells via oxidative stress, which was associated with down regulated Nrf2/HO‐1 pathway, and the mitochondria dependent apoptotic signaling pathway.
Prion diseases are transmissible fatal neurodegenerative disorders spreading between humans and other mammals. The pathogenic agent, prion, is a protease‐resistant, β‐sheet‐rich protein aggregate, ...converted from a membrane protein called PrPC. PrPSc is the misfolded form of PrPC and undergoes self‐propagation to form the infectious amyloids. Since the key hallmark of prion disease is amyloid formation, identifying and studying which segments are involved in the amyloid core can provide molecular details about prion diseases. It has been known that the prion protein could also form non‐infectious fibrils in the presence of denaturants. In this study, we employed a combination of site‐directed nitroxide spin‐labeling, fibril seeding, and electron spin resonance (ESR) spectroscopy to identify the structure of the in vitro‐prepared full‐length mouse prion fibrils. It is shown that in the in vitro amyloidogenesis, the formation of the amyloid core is linked to an α‐to‐β structural transformation involving the segment 160‐224, which contains strand 2, helix 2, and helix 3. This method is particularly suitable for examining the hetero‐seeded amyloid fibril structure, as the unlabeled seeds are invisible by ESR spectroscopy. It can be applied to study the structures of different strains of infectious prions or other amyloid fibrils in the future.
Bubbling O2 into a THF solution of CoII(BDPP) (1) at −90 °C generates an O2 adduct, Co(BDPP)(O2) (3). The resonance Raman and EPR investigations reveal that 3 contains a low spin cobalt(III) ion ...bound to a superoxo ligand. Significantly, at −90 °C, 3 can react with 2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOH) to form a structurally characterized cobalt(III)-hydroperoxo complex, CoIII(BDPP)(OOH) (4) and TEMPO•. Our findings show that cobalt(III)-superoxo species are capable of performing hydrogen atom abstraction processes. Such a stepwise O2-activating process helps to rationalize cobalt-catalyzed aerobic oxidations and sheds light on the possible mechanism of action for Co-bleomycin.
Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH+-PPase), ...the VrH+-PPase–2Pi complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH+-PPases complexes (Pi-, 2Pi- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2Pi-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH+-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of VrH+-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.
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•The pore diameters along the ion translocation pathway of VrH+-PPase of different functional states were determined.•Pyrophosphate hydrolysis and proton pumping coupling efficacies of VrH+-PPase mutants identify the roles of hydrophobic gate and exit channel.•The solvation of the hydrophobic gate of VrH+-PPase was observed during the proton translocation.•Salt-bridge, E225–R562 at the exit channel acts as a latch to regulate ion release of VrH+-PPase.
We report two novel three‐dimensional copper‐benzoquinoid metal–organic frameworks (MOFs), Cu4L3n and Cu4L3 ⋅ Cu(iq)3n (LH4=1,4‐dicyano‐2,3,5,6‐tetrahydroxybenzene, iq=isoquinoline). Spectroscopic ...techniques and computational studies reveal the unprecedented mixed valency in MOFs, formal Cu(I)/Cu(III). This is the first time that formally Cu(III) species are witnessed in metal–organic extended solids. The coordination between the mixed‐valence metal and redox‐non‐innocent ligand L, which promotes through‐bond charge transfer between Cu metal sites, allows better metal‐ligand orbital overlap of the d‐π conjugation, leading to strong long‐range delocalization and semiconducting behavior. Our findings highlight the significance of the unique mixed valency between formal Cu(I) and highly‐covalent Cu(III), non‐innocent ligand, and pore environments of these bench stable Cu(III)‐containing frameworks on multielectron transfer and electrochemical properties.
The first metal–organic frameworks with formally Cu(I)/Cu(III) mixed valency are reported. These bench stable Cu(III)‐containing frameworks coordinated by a non‐innocent ligand are characterized and investigated for multielectron redox properties, unveiling the redox‐active sites at both the copper and organic ligand. This is also the first time that formally Cu(III) species are witnessed in metal–organic extended solids.
Genotoxic stress from environmental pollutants plays a critical role in cytotoxicity. The most abundant nitro-polycyclic aromatic hydrocarbon in environmental pollutants, 1-nitropyrene (1-NP), is ...generated during fossil fuel, diesel, and biomass combustion under sunlight. Macrophages, the key regulators of the innate immune system, provide the first line of defense against pathogens. The toxic effects of 1-NP on macrophages remain unclear. Through a lactate dehydrogenase assay, we measured the cytotoxicity induced by 1-NP. Our results revealed that 1-NP induced genotoxicity also named DNA damage, including micronucleus formation and DNA strand breaks, in a concentration-dependent manner. Furthermore, 1-NP induced p53 phosphorylation and nuclear accumulation; mitochondrial cytochrome c release; caspase-3 and -9 activation and cleavage; and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in a concentration-dependent manner. Pretreatment with the PARP inhibitor, 3-aminobenzamide, significantly reduced cytotoxicity, genotoxicity, and PARP-1 cleavage induced by 1-NP. Pretreatment with the caspase-3 inhibitor, z-DEVD-fmk, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, and caspase 3 activation induced by 1-NP. Pretreatment with the p53 inhibitor, pifithrin-α, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, caspase 3 activation, and p53 phosphorylation induced by 1-NP. We propose that cytotoxicity and genotoxicity induced by 1-NP by PARP-1 cleavage via caspase-3 and -9 activation through cytochrome c release from mitochondria and its upstream p53-dependent pathway in macrophages.
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•1-NP induced cytotoxicity and genotoxicity in RAW264.7 macrophages.•1-NP induced micronucleus formation and DNA strand breaks.•1-NP induced p53 phosphorylation and nuclear accumulation, cytochrome c release, caspase-3 and -9 activation.•1-NP induced PARP-1 cleavage.
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