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Preserved two-photon optical properties of hydrophilic proteins-conjugated quantum dots
摘要： Nonlinear optical properties of colloidal CdSe quantum dots conjugated with proteins were investigated in a wide spectral range with the two-photon excited emission technique using a tunable femtosecond laser system in the range from 700 to 1000 nm. The most signi?cant value of the two-photon absorption cross section σ2 for 2.9 nm size hydrophilic CdSe quantum dot was equal to 4 505 GM, while the two-photon excitation action cross section σ2·QY was found to be 0.101 GM at 900 nm. The nonlinear absorption properties are present as appropriate cross sections normalized per molecular weight which allows comparison of the nonlinear properties of the studied quantum dots with various nanosystems or organic dyes. The properties of these proteins covering hydrophilic colloidal quantum dots can be potentially useful for nonlinear bioimaging.
关键词： Hydrophilization,Semiconducting quantum dots,Nonlinear optical properties,Nanophotonics,Proteins
更新于2025-11-20 15:33:11
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Suppression of Light-Induced Oxidative Stress in the Retina by Mitochondria-Targeted Antioxidant
摘要： Light-induced oxidation of lipids and proteins provokes retinal injuries and results in progression of degenerative retinal diseases, such as, for instance, iatrogenic photic maculopathies. Having accumulated over years retinal injuries contribute to development of age-related macular degeneration (AMD). Antioxidant treatment is regarded as a promising approach to protecting the retina from light damage and AMD. Here, we examine oxidative processes induced in rabbit retina by excessive light illumination with or without premedication using mitochondria-targeted antioxidant SkQ1 (10-(6’-plastoquinonyl)decyltriphenyl-phosphonium). The retinal extracts obtained from animals euthanized within 1–7 days post exposure were analyzed for H2O2, malondialdehyde (MDA), total antioxidant activity (AOA), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) using colorimetric and luminescence assays. Oxidation of visual arrestin was monitored by immunoblotting. The light exposure induced lipid peroxidation and H2O2 accumulation in the retinal cells. Unexpectedly, it prominently upregulated AOA in retinal extracts although SOD and GPx activities were compromised. These alterations were accompanied by accumulation of disulfide dimers of arrestin revealing oxidative stress in the photoreceptors. Premedication of the eyes with SkQ1 accelerated normalization of H2O2 levels and redox-status of lipids and proteins, contemporarily enhancing AOA and, likely, sustaining normal activity of GPx. Thus, SkQ1 protects the retina from light-induced oxidative stress and could be employed to suppress oxidative damage of proteins and lipids contributing to AMD.
关键词： SkQ1,superoxide dismutase,glutathione peroxidase,disulfide dimerization of proteins,visual arrestin,age-related macular degeneration,mitochondria-targeted antioxidant,antioxidant activity,light-induced retinal damage,oxidative stress
更新于2025-09-23 15:23:52
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Sensing of iron(III)-biomolecules by surfactant-free fluorescent copper nanoclusters
摘要： Surfactant-free copper nanoclusters (sf-CuNCs) synthesized in the absence of external stabilizer, could be promising fluorescence biosensors because of the presence of relatively exposed surfaces that may ease coupling with biomolecules. This article shows that sf-CuNCs are effective fluorescence sensor of iron(III)-biomolecules: hemin, cytochrome C (cyt C), and ferritin. Although, sf-CuNCs are found to be weaker sensor of aqueous Fe3+ (Stern-Volmer constant (KSV) 3.0 × 103 M?1 and limit of detection (LOD) up to 2 μM) compared to other capped CuNCs, however, the sensing ability markedly increases in the presence of Fe(III)-biomolecules. KSV (LOD) are 5.3 × 104 M?1 (0.8 μM), 9.0 × 105 M?1 (68 nM), 1.6 × 106 M?1 (16.50 nM), respectively for cyt C, hemin and ferritin. We carried out detailed mechanistic investigations of the quenching process using time-resolved fluorescence, zeta potential and transmission electron microscopy (TEM) measurements. We found that fluorescence quenching is associated with complexation of Fe3+ with CuNCs which changes zeta potential of the sf-CuNCs and leads to aggregation of CuNCs. Moreover, fluorescence quenching, zeta potential and aggregation of CuNC can be reversed by addition of vitamin C which is a very strong chelator and reducing agent of Fe(III). Vitamin C reduces Fe(III) to Fe(II) and promotes disassembly of CuNCs-aggregates to regenerate fluorescence intensity. Since, the sensing is primarily governed by complex formation with Fe3+, absence of ligands on the surface of sf-CuNCs may be detrimental to quenching to aqueous Fe3+ but favors binding with the biomolecules.
关键词： Iron sensing,Copper nanocluster,Proteins,TEM,Fluorescence
更新于2025-09-23 15:23:52
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Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor
摘要： UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.
关键词： intrinsically disordered proteins,plant photoreception,UVR8,native mass spectrometry,ion mobility
更新于2025-09-23 15:23:52
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Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors
摘要： Centrins (CETN1–4) are ubiquitous and conserved EF–hand family Ca2+-binding proteins associated with the centrosome, basal body, and transition zone. Deletion of CETN1 or CETN2 in mice causes male infertility or dysosmia, respectively, without affecting photoreceptor function. However, it remains unclear to what extent centrins are redundant with each other in photoreceptors. Here, to explore centrin redundancy, we generated Cetn3GT/GT single-knockout and Cetn2-/-; Cetn3GT/GT double-knockout mice. Whereas the Cetn3 deletion alone did not affect function, simultaneous ablation of Cetn2 and Cetn3 resulted in attenuated scotopic and photopic electroretinography (ERG) responses in mice at three months of age, with nearly complete retina degeneration at one year. Removal of CETN2 and CETN3 activity from the lumen of the connecting cilium (CC) destabilized the photoreceptor axoneme and reduced the CC length as early as postnatal day 22 (P22). In Cetn2-/-; Cetn3GT/GT double-knockout mice, spermatogenesis-associated 7 (SPATA7), a key organizer of the photoreceptor-specific distal CC, was depleted gradually and CETN1 was condensed to the mid-segment of the CC. Ultrastructural analysis revealed that in this double knockout, the axoneme of the CC expanded radially at the distal end, with vertically misaligned outer segment discs and membrane whorls. These observations suggest that CETN2 and CETN3 cooperate in stabilizing the CC/axoneme structure.
关键词： centriole,connecting cilium,retina degeneration,cilium,retina,electroretinography,Ca2+-binding proteins,photoreceptor,centrin 2/centrin 3 double knockout,centrin
更新于2025-09-23 15:22:29
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Phase transformations of bovine serum albumin: Evidences from Rayleigh-Brillouin light scattering
摘要： Rayleigh‐Brillouin light‐scattering studies of a high‐concentration solution of bovine serum albumin (BSA, pH = 7.45, C = 100 mg/ml) in a sodium phosphate buffer in the temperature range 300–370 K are presented. It is shown that the temperature dependences of relative changes in the Brillouin shift and half width at half maximum and intensity of the Brillouin doublet and intensity of Rayleigh components exhibit anomalies in the vicinity of 335, 345, and 363 K. Analysis of the literature data allowed us to construct a phase diagram showing a sequence of BSA phase transformations in the temperature range from 300 to 375 K and to correlate the anomalies in the scattering spectra to the regions of phase transformations. The anomalies in the behavior of the light‐scattering spectra in the vicinity of Tm = 335 K are attributable to the melting temperature and the formation of BSA self‐assembled aggregates. The formation of a phase with oriented β‐sheet rich mature fibrils at Tm < T < 347 K is confirmed by elastic light scattering experiments. This phase passes into a gel‐like phase in which the aggregates are rearranged and a polymer network, which is finally converted to a gel in the vicinity of 363 K is formed. Thus, the Rayleigh‐Brillouin scattering experiments demonstrated the evolution of the structure, which manifested itself in the anomalous temperature behavior of the intensities of the Rayleigh and Brillouin components in the light‐scattering spectra. The anomalous low‐frequency dynamics of the BSA solution at denaturing and transition into a gel phase was clearly observed in the anomalous Brillouin shift behavior in the vicinity of 335 and 363 К. The sequence of phase transformations in BSA at heating from 300 to 375 K in the phase diagram correlates well with the anomalies observed in the evolution of Rayleigh‐Brillouin light scattering.
关键词： transformation,proteins,aggregation,phase,Brillouin spectroscopy,gelation
更新于2025-09-23 15:22:29
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Extracellular biosynthesis of Cu2-xSe nanocrystallites with photocatalytic activity
摘要： Semiconductor Cu2-xSe nanospheres were successfully biosynthesized based on bioreduction of SeO3 2? into Se2? by the selenite-reducing bacterium, Pantoea agglomerans. The Cu2-xSe nanospheres had excellent crystallinity with a face-centered cubic structure and an average diameter of 80 nm. Composition and oxidation states analysis using X-ray photoelectron spectroscopy and X-ray energy dispersive spectroscopy followed by optical characterization using ultraviolet-visible and Fourier transform infra-red spectroscopy confirmed that the biosynthesized Cu2-xSe nanospheres were capped by proteins. The extracellular proteins which mediated biosynthesis were visualized by excitation–emission matrix fluorescence spectroscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis revealed that the molecular masses of proteins were about 110, 50, 38, 35 and 25 kDa. The biosynthesized Cu2?xSe nanospheres showed an excellent and stable photocatalytical activity under sunlight irradiation in the degradation of methylene blue for four cycles. This study put forward a green and toilless way to manufacture copper selenide nanoparticles using a biological process.
关键词： Biosynthesis,Photocatalytical activity,Cu2-xSe,Extracellular proteins
更新于2025-09-23 15:21:21
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Discovery and Characterization of a Naturally Occurring, Turn-On Yellow Fluorescent Protein Sensor for Chloride
摘要： Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of ?uorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identi?cation and spectroscopic characterization of the yellow ?uorescent protein from the jelly?sh Phialidium sp. (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on ?uorescent protein sensor for chloride. Our results show that chloride binding tunes the pKa of the chromophore Y66 and shifts the equilibrium from the ?uorescent phenolate form to the weakly ?uorescent phenol form. The latter likely undergoes excited-state proton transfer to generate a turn-on ?uorescence response that is pH-dependent. Moreover, anion selectivity and mutagenesis in the chloride binding pocket provide additional evidence for the proposed chloride sensing mechanism. Given these properties, we anticipate that phiYFP, with further engineering, could be a new tool for imaging cellular chloride dynamics.
关键词： phiYFP,mutagenesis,chloride sensor,anion selectivity,turn-on fluorescence,pH-dependent,excitation ratiometric,fluorescent proteins
更新于2025-09-23 15:21:21
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Assessment of functionals for TDDFT calculations of one- and two-photon absorption properties of neutral and anionic fluorescent proteins chromophores
摘要： Performance of DFT functionals with different percentage of exact Hartree-Fock exchange energy (EX) is assessed for recovery of the CC2 reference one- (OPA) and two-photon absorption (TPA) spectra of fluorescent proteins chromophores in vacuo. The investigated DFT functionals, together with their EX contributions are: BLYP (0%), B3LYP (20%), B1LYP (25%), BHandHLYP (50%) and CAM-B3LYP (19% at short range and 65% at long range). Our test set consists of anionic and neutral chromophores as naturally occuring in the fluorescent proteins. For the first time, we compare TDDFT and CC2 methods for higher excited states, than the S1 state, exhibiting relatively large TPA intensity. Our TDDFT results for neutral chromophores reveal an increase of excitation energies as well as TPA and OPA intensities errors, compared to CC2-derived results, as the DFT functional contains less exact exchange. The long-range-corrected CAM-B3LYP functional performs the best, closely followed by BHandHLYP, while BLYP usually significantly underestimates all investigated spectral properties, hence being the worst in reproducing the reference CC2 results. The hybrid B3LYP and B1LYP functionals can be roughly placed in between. We propose that TDDFT may underestimate the TPA intensities for neutral chromophores of fluorescent proteins due to underestimated oscillator strengths between some excited states. In case of anionic chromophores, we find that B3LYP and B1LYP functionals overcome others in terms of reproducing CC2 excitation energies. On the other hand, however, TPA intensity is usually significantly underestimated and in this respect CAM-B3LYP functional seems to be again superior. In contrast to the case of neutral chromophores, it seems that large magnitude of excited-state dipole moment or change in dipole moment upon excitation may be the driving force behind high TPA transition moments.
关键词： fluorescent proteins chromophores,one-photon absorption,DFT functionals,two-photon absorption,TDDFT,CC2
更新于2025-09-23 15:21:21
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Ancient Family of Retinal Proteins Brought to Light “Sight-Unseen”
摘要： Rhodopsins are an ancient class of light-sensing membrane proteins that bind retinylidene chromophores in order to form pigments that absorb visible light. Light absorption triggers isomerization of the chromophore, which is bound covalently within the transmembrane core of the protein, an event that drives protein conformational changes. Type-1 rhodopsins are also known as microbial rhodopsins, which include the well-known light-driven protein pump bacteriorhodopsin found in archeabacteria. Type-2 rhodopsins, or animal rhodopsins, include the sensory rhodopsins used as visual pigments found in most animals, including humans. Both Type-1 and -2 rhodopsins share a canonical seven-transmembrane helical polytopic structure with their amino terminal tails projecting to the topological outside of the cell membrane. Rhodopsins harness light energy to perform a vast array of functions, including pumping ions, gating channel activity, activating coupled enzyme systems, and transducing light sensation in vision. Pushkarev et al. have now reported the discovery of an entirely new group of microbial rhodopsins, opening up the potential for the elucidation of an extensive new class of rhodopsins that they term “heliorhodopsins.”
关键词： heliorhodopsins,retinylidene chromophores,light-sensing,membrane proteins,rhodopsins
更新于2025-09-23 15:21:21