RESUMEN

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.

RESUMEN

Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.

Asunto(s)

RESUMEN

Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.

Asunto(s)

RESUMEN

Phosphorylation, one of the important protein post-translational modifications, is involved in many essential cellular processes. Site-specifical and homogeneous phosphoproteins can be used as probes for elucidating the protein phosphorylation network and as potential therapeutics for interfering their involved biological events. However, the generation of phosphoproteins has been challenging owing to the limitation of chemical synthesis and protein expression systems. Despite the pioneering discoveries in phosphoprotein synthesis, over the past decade, great progresses in this field have also been made to promote the biofunctional exploration of protein phosphorylation largely. Therefore, in this review, we mainly summarize recent advances in phosphoprotein synthesis, which includes five sections: 1) synthesis of the nonhydrolyzable phosphorylated amino acid mimetic building blocks, 2) chemical total and semisynthesis strategy, 3) in-cell and in vitro genetic code expansion strategy, 4) the late-stage modification strategy, 5) nonoxygen phosphoprotein synthesis.

Asunto(s)

RESUMEN

We developed a new responsive peptide hydrogel FmocFFpSC(oNB)-PEG, which could achieve gel formation induced by calcium ions and sequential dissolution stimulated by light. It provides a potential delivery system for the efficient encapsulation of drugs and their controlled release in a spatial and temporal way.

Asunto(s)

RESUMEN

A method is described for surface-enhanced Raman scattering (SERS) discrimination of formaldehyde (FA) and acetaldehyde (AA) in aqueous sample solutions. It is based on the use of a paper strip containing 4-aminothiophenol (Atp)-modified reduced graphene oxide (rGO)/[Ag(NH3)2]+ (rGO/[Ag(NH3)2]+/Atp). The addition of FA or AA induces the conversion of [Ag(NH3)2]+ complex to silver nanoparticles (AgNPs) because of aldehyde-induced silver reduction reaction. The AgNPs possess strong SERS activity. The average interparticle gaps between the AgNPs can be fine-tuned by controlling the experimental conditions, this leading to the formation of optimized SERS hot spots. It is also found that the changes in the spectral shapes and the relative intensity ratio of the bands at 1143 and 1072 cm-1 result from the difference in the pH value of the surrounding solution. This effect enables the selective discrimination of FA and AA. The paper strip can be used as a SERS dipstick and swab for on-site determination of FA or AA in wine and human urine via the differences in the intensity of the SERS peaks. The assay works over a wide range of concentrations (0.45 ng·L-1 to 480 µg·L-1) for FA and AA, and the respective detection limits are 0.15 and 1.3 ng·L-1. Graphical abstract Schematic presentation of the preparation procedure of 4-aminothiophenol (Atp)-modified reduced graphene oxide (rGO)/[Ag(NH3)2]+ hybrid paper and its surface-enhanced Raman scattering discrimination of formaldehyde and acetaldehyde based on silver reduction.

RESUMEN

We report a facile synthetic strategy toward CH2-substituted phosphothreonine mimetics. Herein, inexpensive valine with a directing group was converted into homothreonine via palladium-catalyzed γ-methyl C(sp3)-H bond activation, followed by construction of a phosphorus-carbon bond via the well-developed Appel reaction and Michaelis-Becker reaction with a total yield of 30%. Furthermore, the derived mimetic was applied for solid-phase synthesis of two phosphopeptide inhibitors. This efficient synthesis provides a chance to prepare not only phosphopeptides but also phosphoproteins resistant to phosphatases.

RESUMEN

A fluorescent and surface-enhanced Raman spectroscopy (SERS) dual-mode probe is developed for imaging of intracellular Zn2+ based on N-(2-(bis(pyridine-2-ylmethyl)amino)ethyl)-2-mercaptoacetamide (MDPA) modified gold nanoparticles (MDPA-GNPs). Benefiting from the chelation-enhanced fluorescence (CHEF) between MDPA-GNPs and Zn2+, the fluorescent intensities of MDPA-GNPs are substantially enhanced with the increment of Zn2+ concentrations, which can be clearly observed by the naked eye. Under physiological conditions, the probe exhibits a stable response for Zn2+ from 1 µM to 120 µM, with a detection limit of 0.32 µM in aqueous solutions. The resultant MDPA-GNPs can be used for ultrasensitive SERS detection of Zn2+ because of the strong inter-particle plasmonic coupling generated in the process of Zn2+-triggered MDPA-GNPs self-aggregation, with a low detection limit of 0.28 pM, which is eight order of magnitude lower than the United States Environmental Protection Agency (US EPA)-defined limit (76 µM) in drinkable water. More importantly, the proposed probe can be applied for efficient detection of intracellular Zn2+ with excellent biocompatibility and cellular imaging capability. Therefore, a highly sensitive and selective nanosensor has been demonstrated for both reliable quantitative detection of Zn2+ in aqueous solution and real-time imaging of intracellular Zn2+, suggesting its significant potential utility in bioanalysis and biomedical detection in the future.

Asunto(s)

RESUMEN

A novel and facile synthetic strategy for α,α-difluorinated phosphonate mimetics of phosphoserine/phosphothreonine utilizing rhodium-catalyzed asymmetric hydrogenation was developed. The dehydrogenated substrate ß-difluorophosphonomethyl α-(acylamino)acrylates were first prepared from protected serine/threonine followed by asymmetric hydrogenation using the rhodium-DuPhos catalytic system to generate the chiral center(s). These important phosphonate building blocks were successfully incorporated into phosphatase-resistant peptides, which displayed similar inhibition to the 14-3-3 ζ protein as the parent pSer/pThr peptides.

RESUMEN

This study demonstrates a novel strategy for colorimetric/surface-enhanced Raman scattering (SERS) dual-mode sensing of sulfur dioxide (SO2) by coupling headspace sampling (HS) with paper-based analytical device (PAD). The smart and multifunctional PAD is fabricated with a vacuum filtration method in which 4-mercaptopyridine (Mpy)-modified gold nanorods (GNRs)-reduced graphene oxide (rGO) hybrids (rGO/MPy-GNRs), anhydrous methanol, and starch-iodine complex are immobilized into cellulose-based filter papers. The resultant PAD exhibits a deep-blue color with a strong absorption peak at 600 nm due to the formation of an intermolecular charge-transfer complex between starch and iodine. However, the addition of SO2 induces the Karl Fischer reaction, resulting in the decrease of color and increase of SERS signals. Therefore, the PAD can be used not only as a naked-eye indicator of SO2 changed from blue to colorless but also as a highly sensitive SERS substrates because of the SO2-triggered conversion of Mpy to pyridine methyl sulfate on the GNRs. A distinguishable change in the color was observed at a SO2 concentration of 5 µM by the naked eye, and a detection limit as low as 1.45 µM was obtained by virtue of UV-vis spectroscopy. The PAD-based SERS method is effective over a wide range of concentrations (1 µM to 2000 µM) for SO2, and the detection limit for SO2 is found to be 1 µM. The HS-PAD based colorimetric/SERS method is applied for the determination of SO2 in wine, and the detection results match well with those obtained from the traditional Monier-Williams method. This study not only offers a new method for on-site monitoring of SO2 but also provides a new strategy for designing of paper-based sensing platform for a wide range of field-test applications.

Asunto(s)

RESUMEN

Although graphitic carbon nitride (GCN) has been intensively studied in photocatalytic research, its performance is still hindered by its inherently low photo-absorption and inefficient charge separation. Herein, we report a simple NaF solution treating method to produce fluorinated and alkaline metal intercalated ultrathin GCN with abundant in-plane pores and exposed active edges, and therefore an enhanced number of actives sites. Compared to bulk GCN, NaF treated GCN has a larger specific surface area of 81.2 m2 g-1 and a relatively narrow band gap of 2.60 eV, which enables a 6-fold higher photocatalytic rate of hydrogen evolution.

RESUMEN

This study demonstrates a novel strategy for colorimetric/fluorescent/surface enhanced Raman scattering (SERS) triple-mode sensing of nitrite based on Griess reaction modulated gold nanorods (GNRs)-Azo-gold nanoparticles (GNPs) (GNRs-Azo-GNPs) assembly. The p-aminothiophenol (PATP)-capped GNRs (GNRs@PATP) and 1,8-diaminonaphthalene (DAP)-modified GNPs (GNPs@DAP) are first synthesized through Au-S and Au-N bonds, respectively. Upon being excited at 365nm, the dispersion of GNRs-GNPs (GNRs@PATP and GNPs@DAP) emits cyan fluorescence. Next, the addition of nitrite into the GNRs-GNPs induces the formation of GNRs-Azo-GNPs assembly, resulting in the enhancement of color and decrease of fluorescence. Therefore, the GNRs-Azo-GNPs assembly can not only be used as a naked-eye indicator of nitrite changed from orange-yellow to purple, but also as a highly selective ''fluorescence quenching'' probe due to the fluorescence resonance energy transfer (FRET) between azo-moiety and DAP. The limit of detection (LOD) for nitrite is 0.05µM by colorimetry and 0.01µM by fluorescence. Meanwhile, the GNRs-Azo-GNPs assembly possesses controllable core-satellites nanostructures and enables on-field SERS detection of nitrite with the LOD of 0.8nM. More importantly, the GNRs-GNPs sensing system can not only be used as a paper-based test strip for on-site fast screening of nitrite with a high sensitivity and selectivity, but also as a SERS substrate for reliable quantitative analysis of nitrite. This study offers a new method for on-site visual detection of nitrite in human urine and meat products, as well as provides a strategy for designing multi-mode sensing platform for various applications.

Asunto(s)

RESUMEN

The authors have developed a method for simultaneous quantification of several charged pesticides (as shown for amitrole, simazine, trichlorfon and bisultap). It is based on the use of a reduced graphene oxide-modified screen-printed electrode (RGO-SPE) and combines electrokinetic trapping (EKT) and surface-enhanced Raman scattering (SERS). When a 50 µL droplet containing negatively charged RGO and positively charged gold nanorods is placed on the SPE, the RGO and gold nanorods are selectively attracted on the surface of the SPE during EKT. This leads to the formation of sandwich-type hybrid substrates. The resulting substrates also contain Raman "hot spots" among the high-density gold nanorods. This, along with the excellent adsorption performance of RGO, makes it an excellent SERS substrate for on-site detection of the charged pesticides. The method is highly reproducible and long-term stable. The spot-to-spot variation of the intensity of the SERS is <15%, and the performance of SERS activity is maintained over a period of 6 weeks. The method works over a wide range of concentrations (0.5 nM to 4 µM) for charged pesticides under optimal conditions, with a sub-nanomolar detection limit (at a signal to noise ratio of 3). The EKT-SERS method requires only microliter volumes and takes only minutes for completion. Therefore, the method provides high sensitivity for detection while preserving the selectivity and stability required for reliable quantitative analysis. Graphical abstract A method combining electrokinetic trapping and SERS can be used for simultaneous detection of charged pesticides in a drop of seawater.