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Photosystem II (PSII) is a large membrane protein (∼700 kDa) complex, harboring P680+, the strongest oxidant known in biological systems, which is responsible for driving tyrosine oxidation and ult...

Characterization of RNA–protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA–protein crosslinker (AMT–NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT–NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA–protein interactions in cells.

Enzymes with tyrosine or tryptophan-based radicals catalyze important reactions. Unnatural amino acids are powerful tools in the study and design of these radical enzymes.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

Selenocysteine (Sec), a rare naturally proteinogenic amino acid, is the major form of essential trace element selenium in living organisms. Selenoproteins, with one or several Sec residues, are found in all three domains of life. Many selenoproteins play a role in critical cellular functions such as maintaining cell redox homeostasis. Sec is usually encoded by an in-frame stop codon UGA in the selenoprotein mRNA, and its incorporation in vivo is highly species-dependent and requires the reprogramming of translation. This mechanistic complexity of selenoprotein synthesis poses a big challenge to produce synthetic selenoproteins. To understand the functions of natural as well as engineered selenoproteins, many strategies have recently been developed to overcome the inherent barrier for recombinant selenoprotein production. In this review, we will describe these progresses in selenoprotein production methodology.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and β-arrestin-1 (βarr1). Using this strategy, we monitored the subtle but functionally important conformational change of βarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection., A facile bioconjugation reaction for site-specific protein modification was developed for smFRET measurement, which detected the subtle but important conformational change of the β-arrestin/GPCR complex for the first time.

Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by the clonal expansion and differentiation arrest of leukemic cells in peripheral blood and bone marrow. Though the treatment using cytarabine-based protocol for AML patients with

Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P)+ and ultimatel...

This study introduces a Ge20Sb15Se65 chalcogenide-glass-based photonic crystal fiber (PCF) with high birefringence and nonlinearity and ultra-low confinement loss, in which two elliptical air holes are added in the core. The propagation properties of the proposed PCF are investigated in mid-infrared range (3.0–5.0 μm) using the finite element method (FEM). Simulation results indicate that optimizing structural parameters can increase the birefringence and nonlinear coefficient to a maximum of 3.16 × 10−1 and 5937 W−1km−1 (in y-polarization mode), respectively, a significantly low confinement loss reach 10−14 dB m−1 at 3.0 μm wavelength, there can obtain one zero dispersion wavelength in the study band, and the designed structure can be fabricated using available technology. The designed PCF will have promising application in mid-infrared fiber fields, optical communication, super-continuum generation, precision, and nonlinear optics.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-β2 adrenergic receptor/β-arrestin-1(β-arr1) membrane protein signaling complex, using only 5 μM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of β-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Current studies on improving the separation performance of cyclone separators focus on five aspects: i] geometry-structure optimization, ii] geometry-parameter optimization, iii] operation-paramete...

In this reported study, a new Q criterion was proposed to analyze the vortex in three cyclones that had various inlet structures. The Reynolds stress model (RSM) was used to simulate the gas flow, and the Phase Doppler Particle Analyzer (PDPA) was used to measure the fluid velocity. The results showed that the vortex structure was intuitive according to the new isovortex surface that was obtained using the Q criterion. The isovortex surface was found to be distorted and not distributed around the center axis. With the development of flow, the equivalent diameter of the isovortex surface gradually decreased. The magnitude of the vorticity and the vortex lines of axisymmetrical volute inlet (AVI) structure were more uniform and symmetrical than those found into the single tangential inlet (STI) structure and the single volute inlet (SVI) structure.

Human indoleamine 2,3-dioxygenase 1 (IDO1) has become an increasingly valuable target for cancer immunotherapy because it promotes immune escape by tumor cells. To date, the function of post-translational modifications (PTMs) on IDO1 has not been fully elucidated. Among the many forms of PTMs, it has been identified that three tyrosine sites (Y15, Y345, and Y353) on IDO1 are nitrated and play important roles in catalytic function. Herein, by genetically encoding 3-nitro-l-tyrosine into the tyrosine nitration sites of IDO1, the homogeneous and native nitrated IDO1 have been obtained. It is found that the nitration of different tyrosine sites has different effects on the IDO1 structure and enzyme activity. Nitration at position Y15 has a negligible effect, but nitration at Y345 or Y353 decreases the enzyme activity, especially Y353. Furthermore, these results demonstrate that the regulation of the catalytic function caused by tyrosine nitration is related to perturbation of the protein structure and heme-binding disruption.

Acute myeloid leukemia (AML) is a clinical and genetic heterogeneous disease with a poor prognosis. Recent advances in genomics and molecular biology have immensely improved the understanding of disease. The advantages of syndrome differentiation and treatment are strong selectivity, good curative effect and lesser side effects. In recent years, according to the molecular mechanism of acute myeloid leukemia, many new therapeutic targets have been found. New targets of differentiation therapy in recent years, such as cell cyclin-dependent kinase (CDK2), isocitrate dehydrogenase (IDH1, IDH2), Homeobox genes (HoxA9), Dihy-droorotate dehydrogenase (DHODH) and some others, are reviewed in this article.

Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine’s sp(2) ε-C-H Dona ana replaces it with a C-S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine’s sp(2) ε-C-H bond and D(2)O. Results from QM/MM calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pK(a) of Tyr353 will affect the deuterium-exchange rate. Indeed, the 3,5-difluoro tyrosine containing EanB catalyzes the deuterium exchange reaction with k(ex) of ~10-fold greater than the wild-type EanB (EanB(WT)). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.

The popularization of smart phones has fostered the use of e-hailing apps, which can effectively reduce information asymmetry and provide ease and convenience during travel. Meanwhile, problems such as product homogeneity, slow operation speed, and interface confusion in travel apps also exist, leading to negative user experience. Building on the theory of planned behavior and technology acceptance model, this study examines multiple features of travel apps and their influence on university students’ experience and travel intentions. Findings of the study suggest that, compared to the contents of travel apps, the ease of use seems to have a stronger influence on students’ attitude, perceived behavioral control, and travel intention. The study contributes to the integration of the technology acceptance model and the theory of planned behavior in travel contexts. The findings also offer meaningful practical implications and recommendations on product and service design to relevant stakeholders willing to offer a better travel app user experience.

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay, which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single-atom replacement in comparison to tyrosine. The arPTE-(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12-fold increase in kcat and 3.2-fold enhancement in kcat /KM at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

In addition to nucleic acids, a variety of other biomolecules have also been found on the plasma membrane. Although researchers have realized that RNA has the ability to bind to membrane vesicles in vitro, little is known about whether and how RNA connects to the plasma membrane of the cell. The combination of high-throughput sequencing and in situ labeling methods provides an innovative approach for large-scale identification of subcellular RNAs. Here, we applied the recently published method APEX-seq and identified 75 RNAs related to the plasma membrane, in which lncRNA PMAR72 (plasma membrane-associated RNA AL121772.1) has a considerable affinity with sphingomyelin (SM) and localizes within distinct membrane foci. Our findings will provide some new evidence to elaborate the relationship between RNA and the plasma membrane of mammalian cells.