RESUMEN
Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions.
Asunto(s)
Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana , Multimerización de Proteína , Proteínas de la Membrana/química , Proteínas de la Membrana/análisis , Electroforesis en Gel de Poliacrilamida/métodos , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/análisis , Cromatografía Liquida/métodos , Western Blotting/métodosRESUMEN
In vitro, 19Fâ NMR methodology is preferably selected as a complementary and straightforward method for unveiling the conformations, dynamics, and interactions of biological molecules. Its effectiveness inâ vivo has seen continuous improvement, addressing challenges faced by conventional heteronuclear NMR experiments on structured proteins, such as severe line broadening, low signal-to-noise ratio, and background signals. Herein, we summarize the distinctive advantages of 19Fâ NMR, along with recent progress in sample preparation and applications within the realm of in-cell NMR. Additionally, we offer insights into the future directions and prospects of this methodology based on our understanding.
RESUMEN
In-cell NMR spectroscopy is an effective tool for observing proteins at atomic resolution in their native cellular environment. However, its utility is limited by its low sensitivity and the extensive line broadening caused by nonspecific interactions in the cells, which is even more pronounced in human cells due to the difficulty of overexpressing or delivering high concentrations of isotopically labeled proteins. Here, we present a high-sensitivity tag (wPSP-6F) containing two trifluoromethyl groups that can efficiently label globular proteins with molecular weights in the 6-40â kDa range under mild conditions. This tag allowed us to detect globular proteins in human cells at concentrations as low as 1.0â µM, which would not have been achievable with 15 N or 3-fluorotyrosine labeling. Moreover, we detected conformational changes and interactions of proteins in the cellular environment. The new sensitive 19 F NMR tag may significantly expand the scope of protein NMR in human cells.
Asunto(s)
Imagen por Resonancia Magnética , Proteínas , Humanos , Proteínas/química , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Mechanoluminescence (ML) and room-temperature photophosphorescence (RTP) were achieved in polymorphisms of a triphenylamine derivative with ortho-substitution. This molecular packing-dependent emission afforded crucial information to deeply understand the intrinsic mechanism of different emission forms and the possible packing-function relationship. With the incorporation of solid-state 13 Câ NMR spectra of single crystals, as well as the analysis of crystal structures, the preferred packing modes for ML and/or RTP were investigated in detail, which can guide the reasonable design of organic molecules with special light-emission properties.
RESUMEN
α-Amylase plays a key role in the physiological cycle of the human body; its function is constantly explored and used as an important indicator of some related diseases like acute pancreatitis, acute organophosphorus pesticide poisoning, and anxiety or depression. However, currently, including the assay kit, existing methods suffer from low sensitivity and time consumption or are indirect assays that require the aid of a tool enzyme or inhibitor of competitive substrates; hence, they are not suitable for the low activity and nondestructive sensing of α-amylase in body fluids. A rapid, highly sensitive, and simple direct α-amylase determination in human body fluids is still challenging. In this work, an AIEgen-based small molecule α-amylase sensing system was first established. The probe has no emission signal in aqueous media because of its good solubility, but the insoluble AIE residues can be released after hydrolysis by α-amylase, lighting up fluorescence significantly. In this novel sensing system, the detection limit is calculated to be 0.14 U L-1 in MES buffer with a linear range of 0-45.5 U L-1, having been shortened to 3 min of test time and excellent selectivity to α-amylase compared to other proteins. Moreover, our method is successfully employed to demonstrate the applications in acute pancreatitis diagnosis and psychological stress analysis. The acquisition of this AIE-based method not only provides a simple technique for clinical diagnosis of related diseases but also has a promotional value for the food and pharmaceutical industries.
Asunto(s)
Líquidos Corporales/enzimología , Sondas Moleculares/análisis , Estilbenos/análisis , alfa-Amilasas/análisis , Humanos , Límite de Detección , Análisis Espectral/métodosRESUMEN
A tetraaryl-1,4-dihydropyrrolo-[3,2-b]pyrroles (TAPP) moiety with the combination of two pyrrole rings and four phenyl moieties demonstrated strong electron-donating ability and nonplanar configuration simultaneously. Once incorporated into the organic dyes as a novel electron donor, it was beneficial for the enhancement of light-harvesting ability and suppression of electron recombination in the photovoltaic and photocatalysis systems. With the linkage of tunable conjugated bridges and electron acceptor, the corresponding organic dyes exhibited improved photovoltaic performance in dye-sensitized solar cells and facilitated photocatalytic hydrogen generation with a highest turnover number (TON) of 4337. Through the detailed investigation of relationship between molecular structures and photovoltaic/photocatalysis property, the connection and difference in molecular design for these two systems are well explained, with the aim to promote the application of dye-sensitized technology in various fields.
RESUMEN
In this work, soybean lecithin (LC) was used to modify ß-cyclodextrin (ß-CD) with hydrophobic fat chains to become amphiphilic (LC-CD), and vitamin E (VE) was encapsulated in former modified ß-CD complexes (LC-CD-VE), the new Pickering emulsions stabilized by LC-CD-VE and LC-CD complexes for the delivery of ß-carotene (BC) were created. The surface tension, contact angle, zeta potential, and particle size were used to assess the changes in complexes nanoparticles at various pH values. Furthermore, LC-CD-VE has more promise as Pickering emulsion stabilizer than LC-CD because of the smaller particle size (271.11 nm), proper contact angle (58.02°), and lower surface tension (42.49 mN/m). The interactions between ß-cyclodextrin, soybean lecithin, and vitamin E were confirmed using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and thermogravimetric analysis (TGA). The durability of Pickering emulsions was examined at various volume fractions of the oil phase and concentrations of nanoparticles. Compared to the emulsion stabilized by LC-CD, the one stabilized by LC-CD-VE showed superior storage stability. Moreover, for the delivery of BC, Pickering emulsions stabilized by LC-CD and LC-CD-VE can outperform bulk oil and Tween 80 stabilized emulsions in terms of UV light stability, storage stability, and bioaccessibility. This work could offer fresh perspectives on stabilizer alternatives for Pickering emulsion delivery systems.
Asunto(s)
Ciclodextrinas , Nanopartículas , beta-Ciclodextrinas , Vitamina E/química , Lecitinas , beta Caroteno/química , Glycine max , Emulsiones/química , beta-Ciclodextrinas/química , Excipientes , Digestión , Tamaño de la PartículaRESUMEN
The wide spread of antibiotic-resistant "superbugs" containing New Delhi metallo-ß-lactamase-1 (NDM-1) has become a threat to human health. However, clinically valid antibiotics to treat the superbugs' infection are not available now. Quick, simple, and reliable methods to assess the ligand-binding mode are key to developing and improving inhibitors against NDM-1. Herein, we report a straightforward NMR method to distinguish the NDM-1 ligand-binding mode using distinct NMR spectroscopy patterns of apo- and di-Zn-NDM-1 titrations with various inhibitors. Elucidating the inhibition mechanism will aid the development of efficient inhibitors for NDM-1.
RESUMEN
ß-Arrestins (ßarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in ßarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of ßarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of ßarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different ßarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of ßarr1 that can be intricately modulated during signaling.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismo , Transducción de Señal/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Arrestina beta 2/metabolismo , FosforilaciónRESUMEN
We report the conformation of a newly discovered specific lanthanide ion (Ln3+) binding protein, lanmodulin (LanM), and its interaction with Ln3+ in Escherichia coli cells using the in-cell NMR technique. We found that LanM remains unfolded and fails to bind Ln3+ in Escherichia coli cells due to the abundance of phosphate groups.
Asunto(s)
Elementos de la Serie de los Lantanoides , Escherichia coli , Iones/química , Elementos de la Serie de los Lantanoides/química , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
Thiols play critical roles in regulating biological functions and have wide applications in pharmaceutical and biomedical industries. However, we still lack a general approach for the simultaneous detection of various thiols, especially in complex systems. Herein, we establish a 19F NMR platform where thiols are selectively fused into a novelly designed fluorinated receptor that has two sets of environmentally different 19F atoms with fast kinetics (k 2 = 0.73 mM-1 min-1), allowing us to generate unique two-dimensional codes for about 20 thiols. We demonstrate the feasibility of the approach by reliably quantifying thiol drug content in tablets, discriminating thiols in living cells, and for the first time monitoring the thiol related metabolism pathway at the atomic level. Moreover, the method can be easily extended to detect the activity of thiol related enzymes such as γ-glutamyl transpeptidase. We envision that the versatile platform will be a useful tool for detecting thiols and elucidating thiol-related processes in complex systems.
RESUMEN
α-Amylase, an essential biomarker in pancreas related diseases and perform a major role in carbohydrates metabolism. Hence, monitoring the dynamic changes of α-amylase is crucial for better clinical diagnosis of diseases. However, the existing methods are suffered from low sensitivity, time consumption and indirect assay with aid of tool enzyme or inhibitor of competitive substrates, the rapid and non-destructive sensing of α-amylase in biological samples was highly desired. In this work, a very simple tetraphenylethylene motif and γ-cyclodextrin based supramolecular fluometric sensing system was firstly established. This system has no emission signal in aqueous media for the freely rotation of phenyl rings in the cavity of γ-cyclodextrin, but the AIE residues can be released in to water after the α-amylase hydrolysing γ-cyclodextrin, then turn on the fluorescence. In this system, the detection limit is calculated to be 0.007 U mL-1 in MES buffer with a linear range of 0-0.35â U mL-1 , having excellent selectivity to α-amylase compared to other proteins. At last, our probe can be applied to the quantitative analysis of α-amylase in human serum, showing potential in point of care testing.
Asunto(s)
Colorantes Fluorescentes/química , Estilbenos/química , alfa-Amilasas/metabolismo , gamma-Ciclodextrinas/química , Bacillus/enzimología , Colorantes Fluorescentes/síntesis química , Humanos , Límite de Detección , Saliva/enzimología , Espectrometría de Fluorescencia , alfa-Amilasas/análisis , alfa-Amilasas/sangreRESUMEN
With respect to cyanoacryclic acid as the traditional acceptor and anchoring group in dye-sensitized solar cells, the introduction of stronger electron-deficient groups has greatly expanded the scope of molecular design and increased their efficiencies for organic dyes. In this contribution, benzothiadiazole (BTD) was selected as a representative acceptor to illustrate the influence of its position on the photophysical properties and corresponding photovoltaic performances. Through the insertion of BTD in different positions of the framework with the same composition units and orders, four sensitizers were designed and synthesized. The structure-property relationship demonstrated the preference of the suitable position of an electron acceptor for optimization of the spectra response and interfacial charge transfer. In our system, dye LI-96 with BTD in the middle of the conjugated bridge showed the broadest spectrum and achieved the best photovoltaic performance (8.25%), which may pave a new way to design or optimize the efficient sensitizers by rational design of the acceptor position.
RESUMEN
A novel ladderlike fused-ring donor, dithienocyclopentacarbazole (DTCC) derivative, is used to design and synthesize three novel donor-acceptor-π-acceptor-type organic dyes (C1-C3) via facile direct arylation reactions, in which the DTCC derivative substituted by four p-octyloxyphenyl groups is served as the electron donor and the carboxylic acid group is used as the electron acceptor or anchoring group. To fine-tune the optical, electrochemical, and photovoltaic properties of the three dyes, various auxiliary acceptors, including benzo[2,1,3]thiadiazole (BT), 5,6-difluorobenzo[2,1,3]thiadiazole (DFBT), and pyridal[2,1,3]thiadiazole (PT), are incorporated into the dye backbones. The results indicate that all of the three dyes exhibit strong light-capturing ability in the visible region and obtain relatively high molar extinction coefficients (>31 000 M-1 cm-1) due to their strong charge transfer (CT) from donor to acceptor. Moreover, theoretical model calculations demonstrate fully separated highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels for the three dyes, which is helpful for efficient charge separation and electron injection. Using the three dyes as sensitizers, conventional dye-sensitized solar cells (DSSCs) based on liquid iodide/triiodide redox electrolytes are fabricated. Our results indicate that the BT-containing dye C1 affords the highest power conversion efficiency of up to 6.75%, much higher than that of the DFBT-containing dye C2 (5.40%) and the PT-containing dye C3 (1.85%). To our knowledge, this is the first example reported in the literature where the DTCC unit has been used to develop novel organic dyes for DSSC applications. Our work unambiguously demonstrates that the ladderlike DTCC derivatives are the superb electron-donating blocks for the development of high-performance organic dyes.
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Purely organic materials with the characteristic of room-temperature phosphorescence (RTP) under ambient conditions demonstrate potential benefits in advanced optoelectronic applications. Exploration of versatile and efficient RTP compounds with low prices is full of challenges due to the slow intersystem crossing process and ultrafast deactivation of the active excited states of organic compounds. Here, a series of boron-containing phosphors were found to present RTP with long-lived lifetimes. Among these commercially available and cheap compounds, (4-methoxyphenyl)boronic acid (PBA-MeO) exhibits long-lived RTP, with a lifetime of 2.24 s, which is among the longest lifetimes of single-component small molecules. Our extensive experiments illustrate that both a rigid conformation and expanded conjugation induced by molecular alignment contribute to the persistent RTP. Because of strong intermolecular interactions via hydrogen bonds, these arylboronic acids easily form crystals and are quite appropriate for anti-forgery materials. Subsequently, we develop a precise, speedy and convenient inkjet printing technology for the fabrication of optoelectronic displays. Furthermore, PBA-MeO is used as an additive to feed Bombyx mori silkworms and shows low toxicity over inorganic materials. Our findings may pave a new way for the development of RTP phosphors and promote their use in practical applications.
RESUMEN
Acceptors in traditional dyes are generally designed closed to TiO2 substrate to form a strong electronic coupling with each other (e.g., cyanoacrylic acid) to enhance the electron injection for the high performance of the corresponding solar cells. However, some newly developed dyes with chromophores or main acceptors isolated from anchoring groups also exhibit comparable or even higher performances. To investigate the relatively untouched electronic coupling effect in dye-sensitized solar cells, a relatively precise method is proposed in which the strength is adjusted gradually by changing isolation spacers between main acceptors and anchoring groups to partially control the electronic interaction. After an analysis of 3 different groups of 11 sensitizers, it is inferred that the electronic coupling should be kept at a suitable level to balance the electron injection and recombination. Based on a reference dye LI-81 possessing a cyanoacrylic acid as acceptor and anchoring group, both photocurrent and photovoltage are synergistically improved after the properties of isolation spacers were changed through the adjustment of the length, steric hindrance, and push-pull electronic characteristic. Accordingly, the rationally designed dye LI-87 with an isolation spacer of thiophene ethylene gives an efficiency of 8.54% and further improved to 9.07% in the presence of CDCA, showing a new way to develop efficient sensitizers.